Real-time PCR for quantification of aerobic anoxygenic phototrophic bacteria based on pufM gene in marine environment

Abstract

The abundance of aerobic anoxygenic phototrophic bacteria (AAPB), a new functional group that plays important roles in marine carbon cycling, is determined frequently by infrared epifluorescence microscopic analysis (IREM) or high-performance liquid chromatography (HPLC) based on detecting BChl a (bacteriochlorophyll a) fluorescence signal at 880 nm. Unfortunately, the fluorescence signal is often influenced by environmental variables and physiological state of cell. Here we developed a real-time quantitative PCR (qPCR) assay based on pufM gene to specifically quantify AAPB in marine environments. High specificity and sensitivity for estimation of AAPB abundance were revealed by analysis of amplification products, melting curves and target sequences. The phylogenetic tree indicated that this primer set is suitable for a wide genetic diversity of AAPB, including α-3, α-4 Proteobacteria and clones of unclear taxonomic position. In contrast, no amplicon was obtained from green non-sulphur bacteria and oxygenic phototrophic bacteria such as Cyanobacterial genomic DNA. The melting behavior could indicate predominant phenotypes in AAPB community in addition to validating the products of qPCR. The AAPB was estimated to range from 1.3 × 10 4 cell/ml to 3.4 × 10 5 cell/ml in our 10 tested water samples by this qPCR assay. Further investigations on the abundance distribution of AAPB in marine environments using the qPCR assay may provide new insight into their ecological functions.