Dams-Kozlowska Hanna, Kaplan David L
Tufts University, Department of Biomedical Engineering, 4 Colby St., Rm. 153, Medford, MA 02155, USA.
Appl Environ Microbiol. 2007 Jun;73(12):4020-8. doi: 10.1128/AEM.00401-07. Epub 2007 Apr 20.
Acinetobacter venetianus Rag1 produces an extracellular, polymeric lipoheteropolysaccharide termed apoemulsan. This polymer is putatively produced via a Wzy-dependent pathway. According to this model, the length of the polymer is regulated by polysaccharide-copolymerase (PCP) protein. A highly conserved proline and glycine motif was identified in all members of the PCP family of proteins and is involved in regulation of polymer chain length. In order to control the structure of apoemulsan, defined point mutations in the proline-glycine-rich region of the apoemulsan PCP protein (Wzc) were introduced. Modified wzc variants were introduced into the Rag1 genome via homologous recombination. Stable chromosomal mutants were confirmed by Southern blot analysis. The molecular weight of the polymer was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Five of the eight point mutants produced polymers having molecular weights higher than the molecular weight of the polymer produced by the wild type. Moreover, four of these five polymers had modified biological properties. Replacement of arginine by leucine (R418L) resulted in the most significant change in the molecular weight of the polymer. The R418L mutant was the most hydrophilic mutant, exhibiting decreased adherence to polystyrene, and inhibited biofilm formation. The results described in this report show the functional effect of Wzc modification on the molecular weight of a high-molecular-weight polysaccharide. Moreover, in the present study we developed a genetic system to control polymerization of apoemulsan. The use of selective exogenous fatty acid feeding strategies, as well as genetic manipulation of sugar backbone chain length, is a promising new approach for bioengineering emulsan analogs.
威尼斯不动杆菌Rag1产生一种称为脱辅基乳化聚糖的细胞外聚合脂杂多糖。这种聚合物据推测是通过Wzy依赖性途径产生的。根据该模型，聚合物的长度由多糖共聚合酶（PCP）蛋白调节。在PCP蛋白家族的所有成员中都鉴定出一个高度保守的脯氨酸和甘氨酸基序，其参与聚合物链长的调节。为了控制脱辅基乳化聚糖的结构，在脱辅基乳化聚糖PCP蛋白（Wzc）富含脯氨酸-甘氨酸的区域引入了特定的点突变。通过同源重组将修饰的wzc变体引入Rag1基因组。通过Southern印迹分析确认了稳定的染色体突变体。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析聚合物的分子量。八个点突变体中的五个产生了分子量高于野生型产生的聚合物分子量的聚合物。此外，这五种聚合物中的四种具有改变的生物学特性。用亮氨酸取代精氨酸（R418L）导致聚合物分子量发生最显著的变化。R418L突变体是最亲水的突变体，表现出对聚苯乙烯的粘附性降低，并抑制生物膜形成。本报告中描述的结果显示了Wzc修饰对高分子量多糖分子量的功能影响。此外，在本研究中，我们开发了一种遗传系统来控制脱辅基乳化聚糖的聚合。使用选择性外源脂肪酸喂养策略以及对糖主链长度进行基因操作，是生物工程乳化聚糖类似物的一种有前景的新方法。
