O'Connor Susan M, Coates John D
Department of Microbiology, Southern Illinois University, Carbondale, Illinois 62901, USA.
Appl Environ Microbiol. 2002 Jun;68(6):3108-13. doi: 10.1128/AEM.68.6.3108-3113.2002.
Recent studies in our lab have demonstrated the ubiquity and diversity of microorganisms which couple growth to the reduction of chlorate or perchlorate [(per)chlorate] under anaerobic conditions. We identified two taxonomic groups, the Dechloromonas and the Dechlorosoma groups, which represent the dominant (per)chlorate-reducing bacteria (ClRB) in the environment. As part of these studies we demonstrated that chlorite dismutation is a central step in the reductive pathway of (per)chlorate that is common to all ClRB and which is mediated by the enzyme chlorite dismutase (CD). Initial studies on CD suggested that this enzyme is highly conserved among the ClRB, regardless of their phylogenetic affiliation. As such, this enzyme makes an ideal target for a probe specific for these organisms. Polyclonal antibodies were commercially raised against the purified CD from the ClRB Dechloromonas agitata strain CKB. The obtained antiserum was deproteinated by ammonium sulfate precipitation, and the antigen binding activity was assessed using dot blot analysis of a serial dilution of the antiserum. The titers obtained with purified CD indicated that the antiserum had a high affinity for the CD enzyme, and activity was observed in dilutions as low as 10(-6) of the original antiserum. The antiserum was active against both cell lysates and whole cells of D. agitata, but only if the cells were grown anaerobically with (per)chlorate. No response was obtained with aerobically grown cultures. In addition to D. agitata, dot blot analysis employed with both whole-cell suspensions and cell lysates of several diverse ClRB representing the alpha, beta, and gamma subclasses of Proteobacteria tested positive regardless of phylogenetic affiliation. Interestingly, the dot blot response obtained for each of the ClRB cell lysates was different, suggesting that there may be some differences in the antigenic sites of the CD protein produced in these organisms. In general, no reactions were observed with cells or cell lysates of the organisms closely related to the ClRB which could not grow by (per)chlorate reduction. These studies have resulted in the development of a highly specific and sensitive immunoprobe based on the commonality of the CD enzyme in ClRB which can be used to assess dissimilatory (per)chlorate-reducing populations in environmental samples regardless of their phylogenetic affiliations.
我们实验室最近的研究表明，在厌氧条件下，将生长与氯酸盐或高氯酸盐[（高）氯酸盐]还原相耦合的微生物无处不在且具有多样性。我们鉴定出两个分类群，即脱氯单胞菌属和脱氯体属，它们是环境中主要的（高）氯酸盐还原菌（ClRB）。作为这些研究的一部分，我们证明了亚氯酸盐歧化是（高）氯酸盐还原途径中的关键步骤，这一途径为所有ClRB所共有，且由亚氯酸盐歧化酶（CD）介导。对CD的初步研究表明，无论其系统发育关系如何，该酶在ClRB中高度保守。因此，这种酶成为针对这些微生物的特异性探针的理想靶点。针对从ClRB脱氯单胞菌菌株CKB纯化的CD，通过商业途径制备了多克隆抗体。所得抗血清通过硫酸铵沉淀法进行脱蛋白处理，并使用抗血清系列稀释的斑点印迹分析来评估抗原结合活性。用纯化的CD获得的效价表明，抗血清对CD酶具有高亲和力，在低至原始抗血清10^(-6)的稀释液中仍观察到活性。该抗血清对脱氯单胞菌的细胞裂解物和完整细胞均有活性，但前提是细胞在厌氧条件下以（高）氯酸盐为底物生长。需氧培养的菌液未出现反应。除脱氯单胞菌外，对代表变形菌门α、β和γ亚类的几种不同ClRB的全细胞悬液和细胞裂解物进行斑点印迹分析，结果均呈阳性，无论其系统发育关系如何。有趣的是，对每种ClRB细胞裂解物获得的斑点印迹反应不同，这表明这些生物体中产生的CD蛋白的抗原位点可能存在一些差异。一般来说，与不能通过（高）氯酸盐还原生长的与ClRB密切相关的生物体的细胞或细胞裂解物未观察到反应。这些研究基于ClRB中CD酶的共性，开发出了一种高度特异性和灵敏的免疫探针，可用于评估环境样品中异化（高）氯酸盐还原菌群，无论其系统发育关系如何。
