Development of a real-time quantitative PCR assay for detection and quantification of the marine bacterium...

Alteromonas macleodii is a ubiquitous marine bacterial species found in a variety of habitats that displays both planktonic and particle-associated lifestyles. Transcriptomic studies demonstrate that, even when present at low abundance, it can make significant contributions to biogeochemical cycles, and its specific association with key marine phytoplankton species indicates other ecological roles as well. It has also been shown to be one of the early colonizers of copper-treated marine vessels. There currently exist no rapid, reliable molecular assays for the detection and quantification of A. macleodii from its different environments. We developed a real-time PCR assay, specific to A. macleodii. This assay targets the DNA gyrase B subunit (gyrB) gene, which occurs as a single copy in the genome. The assay possesses an amplification efficiency of 94.3%, with a limit of detection of 2.5 gyrB copies per μL. Assay specificity was validated by melt curve analysis, followed by sequencing of the amplified product. The assay was specific to thirteen A. macleodii strains and did not amplify other marine bacteria, including Roseobacter denitrificans, Silicibacter sp. TM1040, Vibrio coralliilyticus, Vibrio harveyi, and Vibrio alginolyticus. It also did not amplify Alteromonas mediterranea, a close relative that can occur in the same environment as A. macleodii. This assay was used to determine the presence and abundance of A. macleodii from a range of coastal habitats. The assay was also used to monitor the A. macleodii growth in biofilm and planktonic cultures over time in the presence of elevated copper. This assay provides a rapid and reliable means to assess the presence and abundance of a ubiquitous marine bacterium that, even at low abundance, has been shown to make significant contributions to key marine processes.