Study of a two species microbial community by an inferential comparative genomic analysis tool: Spatial Analytical Microbial Imaging

Abstract

Most molecular fingerprinting techniques, including denaturing gradient gel electrophoresis (DGGE) [1], comparative genomic hybridization (CGH) [2], real-time polymerase chain reaction (RT-PCR) [3], destroy community structure and/or cellular integrity, therefore lost the info. of the spatial locus and the in situ genomic copy number of the cells. An alternative technique, fluorescence in situ hybridization (FISH) doesn't require sample disintegration but needs to develop specific markers and doesn't provide info. related to genomic copy number. Here, a microbial analysis tool, Spatial Analytical Microbial Imaging (SAMI), is described. An application was performed with a mixture of Synechocystis sp. PCC 6803 and E. coli K-12 MG1655. The intrinsic property of their genome, reflected by the average fluorescence intensity (AFI), distinguished them in 3D. And their growth rates were inferred by comparing the total genomic fluorescence binding area (GFA) with that of the pure culture standards. A 93% of accuracy in differentiating the species was achieved. •SAMI does not require sample disintegration and preserves the community spatial structure.•It measures the 3D locus of cells within the mixture and may differentiate them according to the property of their genome.•It allows assessment of the growth rate of the cells within the mixture by comparing their genomic copy number with that of the pure culture standards.

Keywords: 3D imaging analysis; 3D locus; Co-culture; Genomic copy number; Inferential comparative genomic copy number; Microbial community analysis; Spatial Analytical Microbial Imaging; Spatiotemporal microbial community.

Graphical abstract

Fig. 1

The controls and SAMI spatial distribution of the two species. (a), (b) The controls represent the true spatial distribution of the two species in the sample by utilizing autofluorescence emitted by Synechocystis sp. PCC 6803. HO and MA stain the nucleic acid of all species. (c), (d) The spatial distribution of the two species via HO and MA nucleic acid staining using the SAMI method.

Fig. 2

3D distribution of relative genomic copy number in the mixture of E. coli K-12 MG1655 and Synechocystis sp. PCC 6803. Blue circles, represent the relatively faster-growth cells, which have larger number of genomic copies than that of the growth standard. Green circles, represent equal-growth cells with equal number of genomic copies as the growth standard. Yellow circles, represent the slower-growth cells with less genomic copy than that of the growth standard. The growth standard can be the culture of oligoploidy, merodiploidy or polyploidy etc. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Percentage distribution of inferential comparative genomic copy number of E. coli K-12 MG1655 in the mixed culture at different time courses as explained in 1.5. The genomic copy number of each cell in the sample is evaluated by comparing its genome size with that of the growth standard with the genomic copy number considered as “1”. The black bars represent the cells with lower genomic copy number than that of the standard, indicating slower-growth cells. The white bars represent the cells with equal genomic copy number as the standard, hence indicating equal growth rate. The doted bars represent the cells with higher genomic copy number than the standard, demonstrating faster-growth cells. The time interval between each phase is three days.

Fig. 4

Percentage distribution of inferential comparative genomic copy number of Synechocystis sp. PCC 6803 in the mixed culture at different time courses. Synechocystis sp. PCC 6803 in this case is closely linked with the cell division cycle and results in substantial losses from the population. The genomic copy number of each cell in the sample is evaluated by comparing the genome size with that of the growth standard with the genomic copy number considered as “1”. The black bars represent the cells with lower genomic copy number than that of the standard, indicating slower-growth cells. The white bars represent the cells with equal genome copy number and growth rate as the standard. The doted bars represent the cells with higher genomic copy number than that of the standard, hence indicating a faster growth rate than the standard. The time interval between each phase was three days.