Discrimination between live and dead cells in bacterial communities from environmental water samples analyzed by 454 pyrosequencing

摘要: The preferential detection of cells with intact membranes by sample treatment propidium monoazide (PMA) in combination PCR amplification is gaining popularity. This study evaluates the effect PMA on 454 pyrosequencing profiles environmental water samples from a canal Amsterdam and seawater (with sediment) left untreated or exposed to elevated temperatures (50, 60, 85°C) for 10 min. Community analysis was based extraction genomic DNA followed 16S rRNA genes using universal bacterial primers. Whereas highest temperature completely suppressed amplification, products other were subjected massively parallel tag sequencing. did not substantially affect sequence non-heated samples, but heat exposure resulted clear difference relative proportions certain groups. significantly more pronounced heated than water. chosen experimental conditions membrane integrity supported BacLight LIVE/DEAD staining flow cytometry, which confirmed an increase uptake iodide high temperatures.