Stephen J R, Chang Y J, Macnaughton S J, Kowalchuk G A, Leung K T, Flemming C A, White D C
Center for Environmental Biotechnology, University of Tennessee, Knoxville, Tennessee 37932-2575, USA.
Appl Environ Microbiol. 1999 Jan;65(1):95-101. doi: 10.1128/AEM.65.1.95-101.1999.
Contamination of soils with toxic metals is a major problem on military, industrial, and mining sites worldwide. Of particular interest to the field of bioremediation is the selection of biological markers for the end point of remediation. In this microcosm study, we focus on the effect of addition of a mixture of toxic metals (cadmium, cobalt, cesium, and strontium as chlorides) to soil on the population structure and size of the ammonia oxidizers that are members of the beta subgroup of the Proteobacteria (beta-subgroup ammonia oxidizers). In a parallel experiment, the soils were also treated by the addition of five strains of metal-resistant heterotrophic bacteria. Effects on nitrogen cycling were measured by monitoring the NH3 and NH4+ levels in soil samples. The gene encoding the alpha-subunit of ammonia monooxygenase (amoA) was selected as a functional molecular marker for the beta-subgroup ammonia oxidizing bacteria. Community structure comparisons were performed with clone libraries of PCR-amplified fragments of amoA recovered from contaminated and control microcosms for 8 weeks. Analysis was performed by restriction digestion and sequence comparison. The abundance of ammonia oxidizers in these microcosms was also monitored by competitive PCR. All amoA gene fragments recovered grouped with sequences derived from cultured Nitrosospira. These comprised four novel sequence clusters and a single unique clone. Specific changes in the community structure of beta-subgroup ammonia oxidizers were associated with the addition of metals. These changes were not seen in the presence of the inoculated metal-resistant bacteria. Neither treatment significantly altered the total number of beta-subgroup ammonia-oxidizing cells per gram of soil compared to untreated controls. Following an initial decrease in concentration, ammonia began to accumulate in metal-treated soils toward the end of the experiment.
有毒金属对土壤的污染是全球军事、工业和采矿场地面临的一个主要问题。生物修复领域特别关注的是选择用于修复终点的生物标志物。在这个微观世界研究中，我们重点研究了向土壤中添加有毒金属混合物（氯化镉、氯化钴、氯化铯和氯化锶）对属于变形菌β亚群的氨氧化菌（β亚群氨氧化菌）的种群结构和大小的影响。在一个平行实验中，土壤还通过添加五株耐金属异养细菌进行处理。通过监测土壤样品中的NH₃和NH₄⁺水平来测量对氮循环的影响。编码氨单加氧酶α亚基（amoA）的基因被选为β亚群氨氧化细菌的功能分子标志物。对从受污染和对照微观世界中回收的amoA的PCR扩增片段的克隆文库进行了8周的群落结构比较。通过限制性消化和序列比较进行分析。还通过竞争性PCR监测了这些微观世界中氨氧化菌的丰度。回收的所有amoA基因片段都与来自培养的亚硝化螺菌的序列聚类。这些包括四个新的序列簇和一个独特的克隆。β亚群氨氧化菌群落结构的特定变化与金属的添加有关。在接种耐金属细菌的情况下未观察到这些变化。与未处理的对照相比，两种处理均未显著改变每克土壤中β亚群氨氧化细胞的总数。在浓度最初下降之后，氨在实验接近尾声时开始在金属处理过的土壤中积累。
