Fluorescence In Situ Hybridization-Flow Cytometry-Cell Sorting-Based Method for Separation and Enrichment of Type I and Type II Methanotroph Populations

Abstract

A fluorescence in situ hybridization-flow cytometry (FISH/FC)-based method was optimized using artificial mixtures of pure cultures of methanotrophic bacteria. Traditional oligonucleotide probes targeting 16S rRNAs of type I (MG84/705 probe) and type II (MA450 probe) methanotrophs were labeled with fluorescein or Alexa fluor and used for FISH, followed by fluorescence-activated FC analysis and cell sorting (FACS). The method resulted in efficient separation of target cells (type I or type II methanotrophs) from the artificial mixtures. The method was then applied for detection and enrichment of type I and type II methanotroph populations from a natural sample, Lake Washington sediment. Cells were extracted from the sediment, fixed, and subjected to FISH/FC/FACS. The resulting subpopulations were analyzed by reverse transcriptase PCR surveys of 16S rRNA, pmoA (encoding a subunit of particulate methane monooxygenase), and fae (encoding formaldehydeactivating enzyme) genes. The functional gene analysis indicated specific separation of the type I and type II methanotroph populations. 16S rRNA gene analysis revealed that type I methanotrophs comprised 59% of the subpopulation separated using the type I-specific probe and that type II methanotrophs comprised 47.5% of the subpopulation separated using the type II-specific probe. Our data indicate that the FISH/FC/FACS protocol described can provide significant enrichment of microbial populations of interest from complex natural communities and that these can be used for genetic tests. We further tested the possibility of direct wholegenome amplification (WGA) from limited numbers of sorted cells, using artificial mixtures of microbes whose genome sequences are known. We demonstrated that efficient WGA can be achieved using 10 4 or more cells separated by 16S rRNA-specific FISH/FC/FACS, while fewer cells resulted in less specific WGA.

Something from (almost) nothing: the impact of multiple displacement amplification on microbial ecology

“…Reducing bacterial DNA template from 10 ng (410 6 genome copies) by 10-and 100-1000-fold reduced representational bias 3-and 6-fold, respectively (Wu et al, 2006). Other studies also showed increased bias with decreased template copy number (Detter et al, 2002;Bergen et al, 2005;Holbrook et al, 2005;Kalyuzhnaya et al, 2006;Neufeld et al, 2008).…”

“…Even partial genomic drafts from single cells provide genetically linked sequences useful for guiding the assembly of metagenomic sequences into discrete genomes (Lasken, 2007). In a proof of principle for community analysis, cells from a defined mixture of three methylotrophic bacteria were separated by flow sorting (Kalyuzhnaya et al, 2006) using 16S rRNA FISH probes that distinguished between these three methylotrophs. When 10 1 , 10 2 and 10 3 cells were collected in each tube and subjected to MDA, 20%, 50% and 95% of resulting DNA sequences correctly associated with the FISH-targeted organism, and none were from the 'contaminating' organisms; the remaining sequences corresponded to nonspecific DNA synthesis generated as an artifact of MDA.…”

Methanogenic System of a Small Lowland Stream Sitka, Czech Republic

“…Aerobic MOB have been detected in a variety of environments, and in some they represent significant fractions of total microbial communities (e.g. Henckel et al 1999;Carini et al, 2005, Trotsenko & Khmelenina 2005, Kalyuzhnaya et al 2006. However, the data on the diversity and activity of methanotrophic communities from the river ecosystems are yet fragmentary.…”

Methods for Whole Cell Detection of Microorganisms

“…Kalyuzhnaya et al (62) used this approach to examine methanotrophic bacteria from lake sediments and Sekar et al (63) used similar methods to investigate the composition of marine bacterioplankton communities. Post-sorting genomic techniques were used in both cases for additional sample characterization (62,63). Food applications of FISH-FC include the use of a Pseudomonas-specific DNA probe to detect this spoilage organism in milk (64) and detection of Salmonella spp.…”