title: Cytometric fingerprinting for analyzing microbial intracommunity structure variation and identifying subcommunity function.
abstract: Functions of complex natural microbial communities are realized by single cells that contribute differently to the overall performance of a community. Usually, molecular and, more recently, deep-sequencing techniques are used for detailed but resource-consuming phylogenetic or functional analyses of microbial communities. Here we present a method for analyzing dynamic community structures that rapidly detects functional (rather than phylogenetic) coherent subcommunities by monitoring changes in cell-specific and abiotic microenvironmental parameters. The protocol involves the use of flow cytometry to analyze elastic light scattering and fluorescent cell labeling, with subsequent determination of cell gate abundance and finally the creation of a cytometric community fingerprint. Abiotic parameter analysis data are correlated with the dynamic cytometric fingerprint to obtain a time-bound functional heat map. The map facilitates the identification of activity hot spots in communities, which can be further resolved by subsequent cell sorting of key subcommunities and concurrent phylogenetic analysis (terminal restriction fragment length polymorphism, tRFLP). The cytometric fingerprint information is based on gate template settings and the functional heat maps are created using an R script. Cytometric fingerprinting and evaluation can be accomplished in 1 d, and additional subcommunity composition information can be obtained in a further 6 d.
authors.affiliation: Department of Bioenergy, Helmholtz Centre for Environmental Research (UFZ), Leipzig, Germany.
authors.forename: Christin
authors.initials: C
authors.lastname: Koch
authors.forename: Susanne
authors.initials: S
authors.lastname: Günther
authors.forename: Adey F
authors.initials: AF
authors.lastname: Desta
authors.forename: Thomas
authors.initials: T
authors.lastname: Hübschmann
authors.forename: Susann
authors.initials: S
authors.lastname: Müller
mesh_terms: D001419:Bacteria / Q000166:cytology / Q000235:genetics; D044822:Biodiversity; D060833:Cellular Microenvironment; D005002:Ethiopia; D005434:Flow Cytometry / Q000379:methods*; D056265:Microbial Interactions; D016133:Polymerase Chain Reaction / Q000379:methods; D012150:Polymorphism, Restriction Fragment Length; D062065:Wastewater / Q000382:microbiology*
chemical_list: D062065:Wastewater