Tuesday, August 3, 2010

PS 43-143: BrdU incorporation-based flow cytometric sorting and subsequent 16s rDNA pyrosequencing identify polyamine-utilizing bacterioplankton in coastal seawater

Xinxin Lv1, Jisha Jacob1, Mary Ann Moran2, Maria Vila-costa2, and Xiaozhen Mou1. (1) Kent State University, (2) university of Georgia

Background/Question/Methods

Polyamines are short-chained aliphatic organic compounds with multiple amino groups. They are ubiquitously distributed in marine systems with and potentially important sources of nitrogen and carbon to marine plankton community. In order to identify specific bacteria taxa that uptake and utilizing free dissolved polyamine compounds in a natural marine environment, we amended water samples from a southeastern U.S coast with single model polyamine compounds, i.e., putrescine and spermidine. Bromodeoxyuridine (BrdU) was also added in water sample to label bacterioplankton cells that actively responded to the addition of polyamines. BrdU-labeling of bacterial cells was in situ immunofluoresently detected and bacterioplankton populations were analyzed using fluorescence-activated cell sorting (FACS). Within 24 hours, addition of each putrescine and spermidine each induced the formation of distinct bacterioplankton populations with elevated BrdU incorporation level (HI). Cells of HBI were separated from the bulk community and subjected to 16S rDNA V6 region amplification and high throughput pyrosequencing.

Results/Conclusions

A total of 8000 partial 16S rDNA sequences were obtained, average ~1300 sequences for each of the duplicated putrescine HI, spermidine HI and unsorted control cells. Putrescine HI and spermidine HI populations were diversely composed of bacteria from a total of over 70 orders. The major taxa (defined as >2% abundance of the population) of the two polyamine HI populations were similar including Actinomycetales (actinobacteria), Flavobacteriales (bacteroidetes), Bacillales (Firmicutes), Rhodobacterales (alphaproteobacteria), Burkholderiales and Methylophilales (betaproteobacteria), Pseudomonadales, Enterobacteriales and Vibrionales (gammaproteobacteria). Rhodobacterales were the most prominent members in both putrescine HI (20%) and spermidine HI (10%), they only accounted for less than 4% in control populations. The rank and relative abundance of other major taxa varied between putrescine and spermidine HI populations. Our result indicated that polyamine utilization is widely distributed among marine bacteria taxa. This finding was in accordance with a recent gene-based study, which found polyamine transporter genes were widely distributed in sequenced marine genomes and metagenomes.