Metaproteomic and metagenomic analyses of defined oceanic microbial populations using microwave cell fixation and flow cytometric sorting.
A major obstacle in the molecular investigation of natural, especially oceanic, microbial cells is their adequate preservation for further land-based molecular analyses. Here, we examined the use of microwaves for cell fixation before high-speed flow cytometric sorting to define the metaproteomes and metagenomes of key microbial populations. The microwave fixation procedure was established using cultures of Synechococcus cyanobacteria, the photosynthetic eukaryote Micromonas pusilla and the gammaproteobacterium Halomonas variabilis. Shotgun proteomic analyses showed that the profile of microwave-fixed and -unfixed Synechococcus sp. WH8102 cells was the same, and hence proteome identification of microwave-fixed sorted cells by nanoLC-MS/MS is possible. Microwave-fixed flow-sorted Synechococcus cells can also be successfully used for whole-genome amplification and fosmid library construction. We then carried out successful metaproteomic and metagenomic analyses of microwave-fixed Synechococcus cells flow sorted from concentrates of microbial cells, collected in the North Atlantic Ocean. Thus, the microwave fixation procedure developed appears to be useful for molecular studies of microbial populations in aquatic ecosystems.
Mary
Isabelle
I
National Oceanography Centre, Southampton, UK. isabelle.mary@univ-bpclermont.fr
Oliver
Anna
A
Skipp
Paul
P
Holland
Ross
R
Topping
Juliette
J
Tarran
Glen
G
Scanlan
David J
DJ
O'Connor
C David
CD
Whiteley
Andrew S
AS
Burkill
Peter H
PH
Zubkov
Mikhail V
MV
D002853:Chromatography, Liquid; D005240:Feasibility Studies; D005434:Flow Cytometry / Q000379:methods*; D016680:Genome, Bacterial; D056186:Metagenomics / Q000379:methods*; D008872:Microwaves; D009792:Oceans and Seas; D040901:Proteomics / Q000379:methods*; D046940:Synechococcus / Q000145:classification* / Q000235:genetics; D053719:Tandem Mass Spectrometry