Detection and distribution of ammonia-oxidizing bacteria of coastal Georgia waters using molecular-based methods

Abstract

An in situ PCR/hybridization technique was developed to detect the amoA gene in marine ammonia-oxidizing bacteria. A degenerate primer set was used to amplify a region of the amoA functional gene that is common to a range of nitrifying bacteria. Concurrently, FISH techniques were utilized to detect a range of ammonia-oxidizers taxonomically via 16S rRNA homology to an oligonucleotide probe. Samples of coastal Georgia seawater were collected and size fractionated to retain organisms <1.0µm on 0.22µm filters. Filter-adhered bacterial cells were subjected to in situ PCR followed by in situ hybridization with a fluorescently-labeled internal probe. Replicate samples were subjected to 16S rRNA FISH. Filters were examined with epifluoresence microscopy to enumerate both total bacteria and ammonia-oxidizing bacteria. The percentage of AOB detected in the environment ranged from 3.4%-60.0% functionally and 1.2%-23.1% phylogenetically.