Flourishing deep-sea AAP bacteria detected by flow cytometric sorting and molecular analysis

Dajun Qiu,Liangmin Huang,Senjie Lin,Xin Liu,Yiguo Hong

doi:10.1371/journal.pone.0218753

Abstract

Pigmented bacteria cells, including aerobic anoxygenic phototrophic (AAP) bacteria, contribute significantly to secondary production and aquatic carbon cycling but their distribution in the deep sea is still not well understood, especially in the South China Sea. In this study, microscopic, flow cytometric, and molecular analyses were carried out to investigate the abundance and diversity of AAP bacteria at seven stations in the South China Sea. The results revealed the existence of bacteriochlorophyll-containing bacteria below 500 m from two of seven stations. Flow cytometric analysis detected red and infra-red fluorescence under blue (488 nm) light excitation from fluorescent cells. Blue light-excited red fluorescence of these cells from the 1000 m depth at station E403 were verified using epifluorescence microscopy. Based on fluorescence and side scatter features, fluorescent cells were sorted and subjected to molecular analysis. DNA was extracted from these sorted cells from both stations for PCR amplification using 16S rDNA primers. Sequencing of the PCR products showed that the sorted cells from the 1000 m depth at station E403 belonged to the genus Porphyrobacter. The cell population sorted from 500 m at station E703 contained Sphingomonas and a Methylobacterium-like taxon. All these three taxa belong to aerobic anoxygenic phototrophic alpha-proteobacteria. Using flow cytometric analysis, we found that the abundance of Porphyrobacter sp. at 1000 m was 2.71–2.95×104 cells mL-1 whereas cell counts of Sphingomonas sp. and Methylobacterium at 500 m were about 3.75–4.12×105 cells mL-1. These results indicate that albeit not ubiquitous in deep water, bacteriochlorophyll-containing bacteria can be abundant in the deep-sea aphotic zone.

Highlights

The studies of pigment-containing, noncyanobacterial, phototrophic, bacteria have been increasing in the last decades [1,2,3,4,5,6,7,8,9,10,11,12]
These pigmented cells were re-analyzed under the FACSAria cytometer and were found to emit dim infra-red fluorescence at 780/60 nm (Fig 2B), a characteristics of bacteriochlorophyll [25] and cyanobacteria [26]
Bacteriochlorophyll-containing bacteria were detected at two of the seven investigated deep sea stations in the South China Sea. 16S rDNA analysis revealed that the three different lineages of pigmented bacteria at these stations were closely related to Porphyrobacter sanguineum, Sphingomonas sp., and Methylobacterium sp., which belong to different subclasses of Alphaproteobacteria, with the first two in the α-4 subgroup and the last in the α-2 subgroup

Summary

Introduction

The studies of pigment-containing, noncyanobacterial, phototrophic, bacteria have been increasing in the last decades [1,2,3,4,5,6,7,8,9,10,11,12]. Deep-sea AAP bacteria environments and usually show orange, red, pink, brown, or yellow colors due to the presence of various carotenoids [8]. These organisms are usually in low abundance [7,13], but on average their cell sizes are two times larger than regular heterotrophic bacteria in oligotrophic waters [14]. AAP bacteria are capable of utilizing light and organic matter to acquire energy [1]. Kolber et al (2001) found a wide distribution of bacteriochlorophyll-containing, nonphotosynthetic bacteria in surface water, which acquire energy through the oxidation of organic matter [3]. We detected a variety of bacteria that contained bacteriochlorophyll at the depths of 500 and 1000 m

Methods

Results

Discussion

Conclusion