Department of Microbiology, Cornell University, Ithaca, NY 14853, USA.
Environ Microbiol. 2012 Jun;14(6):1544-57. doi: 10.1111/j.1462-2920.2012.02743.x. Epub 2012 Apr 13.
Polaromonas naphthalenivorans strain CJ2 is a Gram-negative betaproteobacterium that was identified, using stable isotope probing in 2003, as a dominant in situ degrader of naphthalene in coal tar-contaminated sediments. The sequenced genome of strain CJ2 revealed several genes conferring nitrogen fixation within a 65.6 kb region of strain CJ2's chromosome that is absent in the genome of its closest sequenced relative Polaromonas sp. strain JS666. Laboratory growth and nitrogenase assays verified that these genes are functional, providing an alternative source of nitrogen in N-free media when using naphthalene or pyruvate as carbon sources. Knowing this, we investigated if nitrogen-fixation activity could be detected in microcosms containing sediments from the field site where strain CJ2 was isolated. Inducing nitrogen limitation with the addition of glucose or naphthalene stimulated nitrogenase activity in amended sediments, as detected using the acetylene reduction assay. With the use of fluorescence microscopy, we screened the microcosm sediments for the presence of active strain CJ2 cells using a dual-labelling approach. When we examined the carbon-amended microcosm sediments stained with both a strain CJ2-specific fluorescent in situ hybridization probe and a polyclonal fluorescently tagged antibody, we were able to detect dual-labelled active cells. In contrast, in sediments that received no carbon addition (showing no nitrogenase activity), no dual-labelled cells were detected. Furthermore, the naphthalene amendment enhanced the proportion of active strain CJ2 cells in the sediment relative to a glucose amendment. Field experiments performed in sediments where strain CJ2 was isolated showed nitrogenase activity in response to dosing with naphthalene. Dual-label fluorescence staining of these sediments showed a fivefold increase in active strain CJ2 in the sediments dosed with naphthalene over those dosed with deionized water. These experiments show that nitrogen fixation may play an important role in naphthalene biodegradation by strain CJ2 and contribute to its ecological success.
萘降解菌 CJ2 是一种革兰氏阴性的β变形菌，于 2003 年通过稳定同位素探测被鉴定为煤焦油污染沉积物中萘的优势原位降解菌。CJ2 菌株的测序基因组揭示了几个基因，这些基因在 CJ2 染色体的 65.6kb 区域内赋予了固氮能力，而在其最接近的测序相关菌——假单胞菌属 JS666 菌株的基因组中则不存在这一区域。实验室生长和氮酶测定验证了这些基因是功能性的，当使用萘或丙酮酸作为碳源时，它们为无氮培养基中提供了一种替代的氮源。考虑到这一点，我们研究了在含有从分离出 CJ2 菌株的野外地点的沉积物的微宇宙中是否可以检测到固氮活性。通过添加葡萄糖或萘来诱导氮限制，刺激了添加沉积物中的氮酶活性，这可以通过乙炔还原测定法检测到。通过荧光显微镜，我们使用双标记方法筛选了微宇宙沉积物中是否存在活性 CJ2 细胞。当我们用 CJ2 菌株特异性荧光原位杂交探针和多克隆荧光标记抗体双重标记来检查添加碳的微宇宙沉积物时，我们能够检测到双标记的活性细胞。相比之下，在没有添加碳的沉积物中（没有检测到氮酶活性），没有检测到双标记细胞。此外，萘的添加增强了相对于葡萄糖添加的沉积物中活性 CJ2 细胞的比例。在分离出 CJ2 菌株的沉积物中进行的现场实验表明，萘处理会导致氮酶活性。对这些沉积物进行的双重荧光染色显示，与用去离子水处理的沉积物相比，用萘处理的沉积物中活性 CJ2 菌株增加了五倍。这些实验表明，氮固定可能在 CJ2 菌株的萘生物降解中发挥重要作用，并有助于其生态成功。
