Ramaiah Nagappa, Chun Jongsik, Ravel Jacques, Straube William L., Hill Russell T., Colwell Rita R.
Center of Marine Biotechnology, University of Maryland Biotechnology Institute, 701 East Pratt Street, Baltimore, MD 21202, USA.
FEMS Microbiol Ecol. 2000 Jul 1;33(1):27-34. doi: 10.1111/j.1574-6941.2000.tb00723.x.
A 745-bp luxA fragment was amplified from Vibrio harveyi (UM 1503), radiolabeled, and used as a probe to detect and quantify luxA genotypes in culturable bacterial populations from the Chesapeake Bay. DNA samples from 53 reference strains were also examined for this gene. The luxA-positive bacteria comprised from 0-6% of the culturable heterotrophic bacterial community in samples from the Bay. Only those reference strains known to be luminescent contained the luxA gene, as indicated by PCR. Results in all cases were confirmed by PCR of DNA extracts and Southern hybridization analyses, using an internal probe for confirmation of luxA amplification products. Sequence analysis of luxA genes from three nonluminescent bacteria isolated from the Chesapeake Bay indicated little or no differences when compared with luxA sequences from known marine luminescent bacterial species. These three Chesapeake Bay strains and other luxA-positive strains were tested with a luminometer and confirmed to be nonluminescent. All of over 7800 bacterial colonies enumerated during this study from Chesapeake Bay samples were non-visibly luminescent. Our results indicate that luxA-positive bacteria isolated from the Chesapeake Bay are not generally luminescent on phenotypic examination, implying that gene probe techniques are required for examining luxA gene distribution in microbial populations present in environmental samples.
从哈维氏弧菌（UM 1503）中扩增出一段745个碱基对的luxA片段，进行放射性标记，用作探针来检测和定量切萨皮克湾可培养细菌群体中的luxA基因型。还对53株参考菌株的DNA样本进行了该基因检测。在来自该海湾的样本中，luxA阳性细菌占可培养异养细菌群落的0%至6%。如PCR检测所示，只有那些已知会发光的参考菌株含有luxA基因。所有结果均通过DNA提取物的PCR和Southern杂交分析得到证实，使用内部探针来确认luxA扩增产物。对从切萨皮克湾分离出的三株非发光细菌的luxA基因进行序列分析，结果表明，与已知海洋发光细菌物种的luxA序列相比，差异很小或没有差异。用光度计对这三株切萨皮克湾菌株和其他luxA阳性菌株进行检测，证实它们不发光。在这项研究中，对来自切萨皮克湾样本的7800多个细菌菌落进行计数，所有菌落均无明显发光现象。我们 的结果表明，从切萨皮克湾分离出的luxA阳性细菌在表型检测中通常不发光，这意味着需要基因探针技术来检测环境样本中微生物群体的luxA基因分布。
