Rockne Karl J, Strand Stuart E
Department of Civil and Materials Engineering, University of Illinois-Chicago, Chicago, Illinois 60607-7023, USA.
J Environ Sci Health A Tox Hazard Subst Environ Eng. 2003 Sep;38(9):1877-87. doi: 10.1081/ese-120022886.
Type II alpha proteobacteria methanotrophs are capable of a wide range of cometabolic transformations of chlorinated solvents and polycyclic aromatic hydrocarbons (PAHs), and this activity has been exploited in many terrestrial bioremediation systems. However, at present, all known obligately marine methanotrophic isolates are Type I gamma proteobacteria which do not have this activity to the extent of Type II methanotrophs. In previous work in our laboratory, determining the presence of Type II alpha proteobacteria methanotrophs in marine enrichment cultures that co-metabolized PAHs required a more sensitive assay. 16S rDNA PCR primers were designed based on oligonucleotide probes for serine pathway methanotrophs and serine pathway methylotrophs with an approximate amplification fragment size of 870 base pairs. Comparison of the primers using double primer BLAST searches in established nucleotide databases showed potential amplification with all Methylocystis and Methylosinus spp., as well as potential amplification with Methylocella palustrus. DNA from Methylosinus trichosporium OB3b, a Type II methanotroph, amplified with the primers with a fragment size of approximately 850 base pairs, whereas DNA extracted from Methylomonas methanica, a Type I methanotroph, did not. The primers were used to amplify DNA extracted from two marine methanotrophic enrichment cultures: a low nitrogen/low copper enrichment to select for Type II methanotrophs and a high nitrogen/high copper enrichment to select for Type I methanotrophs. Although DNA from both cultures amplified with the PCR primers, amplification was stronger in cultures that were specifically enriched for Type II methanotrophs, suggesting the presence of higher numbers of Type II methanotrophs. These results provide further evidence for the existence of Type II marine methanotrophs, suggesting the possibility of exploiting cometabolic activity in marine systems.
II型α-变形菌纲甲烷营养菌能够对氯化溶剂和多环芳烃（PAHs）进行广泛的共代谢转化，这种活性已在许多陆地生物修复系统中得到应用。然而，目前所有已知的专性海洋甲烷营养菌分离株都是I型γ-变形菌纲，它们没有II型甲烷营养菌那样的活性。在我们实验室之前的工作中，要确定共代谢PAHs的海洋富集培养物中II型α-变形菌纲甲烷营养菌的存在，需要更灵敏的检测方法。基于针对丝氨酸途径甲烷营养菌和丝氨酸途径甲基营养菌的寡核苷酸探针设计了16S rDNA PCR引物，扩增片段大小约为870个碱基对。在已建立的核苷酸数据库中使用双引物BLAST搜索对引物进行比较，结果表明所有甲基孢囊菌属和甲基弯曲菌属物种都可能被扩增，以及沼泽甲基小菌也可能被扩增。来自II型甲烷营养菌嗜铁甲基弯曲菌OB3b的DNA用引物扩增后片段大小约为850个碱基对，而来自I型甲烷营养菌甲烷单胞菌的DNA则未被扩增。这些引物被用于扩增从两种海洋甲烷营养菌富集培养物中提取的DNA：一种是低氮/低铜富集培养物以筛选II型甲烷营养菌，另一种是高氮/高铜富集培养物以筛选I型甲烷营养菌。尽管两种培养物的DNA都能用PCR引物扩增，但在专门富集II型甲烷营养菌的培养物中扩增更强，这表明存在更多数量的II型甲烷营养菌。这些结果为II型海洋甲烷营养菌的存在提供了进一步证据，表明在海洋系统中利用共代谢活性的可能性。
