Discrimination between live and dead cellsin bacterial communities from environmental water samples analyzed by 454 pyrosequencing.
The preferential detection of cells with intact membranes by sample treatment with propidium monoazide (PMA) in combination with PCR amplification is gaining in popularity. This study evaluates the effect of PMA on 454 pyrosequencing profiles of environmental water samples from a canal in Amsterdam and seawater (with sediment) left untreated or exposed to elevated temperatures (50, 60, or 85 °C) for 10 min. Community analysis was based on the extraction of genomic DNA followed by PCR amplification of 16S rRNA genes using universal bacterial primers. Whereas the highest temperature in combination with PMA treatment completely suppressed PCR amplification, PCR products from the other samples were subjected to massively parallel tag sequencing. PMA treatment did not substantially affect the sequence profiles of non-heated samples, but heat exposure resulted in a clear difference in the relative proportions of certain groups. This difference was significantly more pronounced in heated seawater than in heated canal water. The effect of the chosen experimental conditions on the membrane integrity of cells was supported by BacLight LIVE/DEAD staining in combination with flow cytometry, which confirmed an increase in the uptake of propidium iodide in samples exposed to high temperatures.
Nocker
Andreas
A
TNO Quality of Life, Business Unit Food and Biotechnology Innovations, Microbial Genomics Group, Zeist, Netherlands.
Richter-Heitmann
Tim
T
Montijn
Roy
R
Schuren
Frank
F
Kort
Remco
R
D001386:Azides / Q000378:metabolism; D001419:Bacteria / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001431:Bacteriological Techniques / Q000379:methods*; D002462:Cell Membrane / Q000378:metabolism; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D006358:Hot Temperature; D056186:Metagenomics / Q000379:methods*; D050296:Microbial Viability; D009426:Netherlands; D011419:Propidium / Q000031:analogs & derivatives / Q000378:metabolism; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods; D013194:Staining and Labeling / Q000379:methods; D014871:Water Microbiology
D001386:Azides; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S; C533957:propidium monoazide; D011419:Propidium