Cheng Q, Yu M C, Reeves A R, Salyers A A
Department of Microbiology, University of Illinois, Urbana 61801, USA.
J Bacteriol. 1995 Jul;177(13):3721-7. doi: 10.1128/jb.177.13.3721-3727.1995.
Bacteroides thetaiotaomicron can utilize a variety of polysaccharides, including charged mucopolysaccharides such as chondroitin sulfate (CS) and hyaluronic acid (HA). Since the enzymes (chondroitin lyases I and II) that catalyze the first step in breakdown of CS and HA are located in the periplasm, we had proposed that the first step in utilization of these polysaccharides was binding to one or more outer membrane proteins followed by translocation into the periplasm, but no such outer membrane proteins had been shown to play a role in CS or HA utilization. Previously we have isolated a transposon-generated mutant, CS4, which was unable to grow on CS or HA but retained the ability to grow on disaccharide components of CS. This phenotype suggested that the mutation in CS4 either blocked the transport of the mucopolysaccharides into the periplasmic space or blocked the depolymerization of the mucopolysaccharides into disaccharides. We have mapped the CS4 mutation to a single gene, csuF, which is capable of encoding a protein of 1,065 amino acids and contains a consensus signal sequence. Although CsuF had a predicted molecular weight and pI similar to those of chondroitin lyases, it did not show significant sequence similarity to the Bacteroides chondroitin lyase II, a Proteus chondroitin ABC lyase, or two hyaluronidases from Clostridium perfringens and Streptococcus pyogenes, nor was any CS-degrading enzyme activity associated with csuF expression in Bacteroides species or Escherichia coli. The deduced amino acid sequence of CsuF exhibited features suggestive of an outer membrane protein. We obtained antibodies to CsuF and demonstrated that the protein is located in the outer membrane. This is the first evidence that a nonenzymatic outer membrane protein is essential for utilization of CS and HA.
多形拟杆菌能够利用多种多糖，包括带电荷的粘多糖，如硫酸软骨素（CS）和透明质酸（HA）。由于催化CS和HA分解第一步的酶（软骨素裂解酶I和II）位于周质中，我们曾提出利用这些多糖的第一步是与一种或多种外膜蛋白结合，然后转运到周质中，但尚未有外膜蛋白被证明在CS或HA利用中发挥作用。此前我们分离出一个转座子产生的突变体CS4，它无法在CS或HA上生长，但保留了在CS二糖成分上生长的能力。这种表型表明CS4中的突变要么阻断了粘多糖向周质空间的转运，要么阻断了粘多糖解聚为二糖的过程。我们已将CS4突变定位到一个单一基因csuF，该基因能够编码一个含有1065个氨基酸的蛋白质，并包含一个共有信号序列。尽管CsuF的预测分子量和pI与软骨素裂解酶相似，但它与多形拟杆菌软骨素裂解酶II、奇异变形杆菌软骨素ABC裂解酶或产气荚膜梭菌和化脓性链球菌的两种透明质酸酶没有显著的序列相似性，在多形拟杆菌属或大肠杆菌中，也没有任何与csuF表达相关的CS降解酶活性。CsuF推导的氨基酸序列显示出提示外膜蛋白的特征。我们获得了针对CsuF的抗体，并证明该蛋白位于外膜中。这是首个证据表明非酶性外膜蛋白对于CS和HA的利用至关重要。
