Abstract
BackgroundThe study of bacterial species interactions in a mixed-species community can be facilitated by transcriptome analysis of one species in the community using cDNA microarray technology. However, current applications of microarrays are mostly limited to single species studies. The purpose of this study is to develop a method to separate one species, Escherichia coli as an example, from mixed-species communities for transcriptome analysis.ResultsE. coli cells were separated from a dual-species (E. coli and Stenotrophomonas maltophilia) community using immuno-magnetic separation (IMS). High recovery rates of E. coli were achieved. The purity of E. coli cells was as high as 95.0% separated from suspended mixtures consisting of 1.1 - 71.3% E. coli, and as high as 96.0% separated from biofilms with 8.1% E. coli cells. Biofilms were pre-dispersed into single-cell suspensions. The reagent RNAlater (Ambion, Austin, TX) was used during biofilm dispersion and IMS to preserve the transcriptome of E. coli. A microarray study and quantitative PCR confirmed that very few E. coli genes (only about eight out of 4,289 ORFs) exhibited a significant change in expression during dispersion and separation, indicating that transcriptional profiles of E. coli were well preserved.ConclusionsA method based on immuno-magnetic separation (IMS) and application of RNAlater was developed to separate a bacterial species, E. coli as an example, from mixed-species communities while preserving its transcriptome. The method combined with cDNA microarray analysis should be very useful to study species interactions in mixed-species communities.
Highlights
The study of bacterial species interactions in a mixed-species community can be facilitated by transcriptome analysis of one species in the community using cDNA microarray technology
The recovery rate dropped to 59.8% for samples with ten-fold higher cells (1.9 × 109 CFU/ml), which may have exceeded the capacity of separation columns used in immuno-magnetic separation (IMS) (Figure 1)
The results demonstrated the feasibility of using IMS to separate E. coli cells from biofilms
Summary
The study of bacterial species interactions in a mixed-species community can be facilitated by transcriptome analysis of one species in the community using cDNA microarray technology. The purpose of this study is to develop a method to separate one species, Escherichia coli as an example, from mixed-species communities for transcriptome analysis. Previous studies have shown that species interactions play an important role in the development, composition, structure and function of microbial consortia in biofilms as well as in suspended growth communities [3,4,5]. Identification of relevant genes is an important step toward the elucidation of the molecular mechanisms of Separating the target species from other community members before extracting RNA could be an approach in minimizing cross hybridization on microarrays. Isolated cells have not been considered for cDNA microarray analysis
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