Catch me if you can: capturing microbial community transformation by extracellular DNA using Hi-C sequencing

Abstract

The transformation of environmental microorganisms by extracellular DNA is an overlooked mechanism of horizontal gene transfer and evolution. It initiates the acquisition of exogenous genes and propagates antimicrobial resistance alongside vertical and conjugative transfers. We combined mixed-culture biotechnology and Hi-C sequencing to elucidate the transformation of wastewater microorganisms with a synthetic plasmid encoding GFP and kanamycin resistance genes, in the mixed culture of chemostats exposed to kanamycin at concentrations representing wastewater, gut and polluted environments (0.01-2.5-50-100 mg L^-1). We found that the phylogenetically distant Gram-negative Runella (102 Hi-C links), Bosea (35), Gemmobacter (33) and Zoogloea (24) spp., and Gram-positive Microbacterium sp. (90) were transformed by the foreign plasmid, under high antibiotic exposure (50 mg L^-1). In addition, the antibiotic pressure shifted the origin of aminoglycoside resistance genes from genomic DNA to mobile genetic elements on plasmids accumulating in microorganisms. These results reveal the power of Hi-C sequencing to catch and surveil the transfer of xenogenetic elements inside microbiomes.

Keywords: Antibiotic resistance; Hi-C sequencing; Mixed cultures; Plasmids; Transformation.

Fig. 1

Schematic representation of the set containing two continuously stirred bioreactors (chemostats). The control chemostat was spike with only kanamycin. In the test chemostat, kanamycin and the synthetic plasmid (pBAV1K-T5-GFP) were spiked daily. This experimental setting was embedded in a biosafety level II laboratory, where samples had to be extracted via a sterile tube welder and handled under sterility in a laminar flow cabinet

Fig. 2

Schematic representation of the Hi-C deconvolution process. Plasmids were cross-linked to chromosomal DNA inside microorganisms of the microbial community with formaldehyde before cell lysis. DNA extract was digested enzymatically, biotinylated, ligated, and purified. To generate the Hi-C library, the resultant fraction was sequenced and used to create Hi-C links that helped deconvolute contigs into genome clusters, including chromosomes and plasmids

Fig. 3

Evolution of biomass in the control and test chemostats. Daily-averaged values for volatile suspended solids (VSS) from both reactor control and reactor with free-floating plasmid over the whole operation time the experiment was conducted (45 days). Kanamycin concentrations are displayed as background-colored sections: 0.01, 2.5, 50, 100 mg L⁻¹

Fig. 4

Microbial community profiles in the control and test chemostats. a Heatmaps from 16SrRNA amplicon sequencing showing bacterial family compositions across the operation in both chemostats grouped per kanamycin concentration (0, 0.01, 2.5, 50, 100 mg L⁻¹ with spiked plasmid and 100 mg L⁻¹—(II) without spiked plasmid). The different color intensities represent the relative bacterial family abundance in each population. RC Reactor Control without spiked plasmid. RT Reactor Test with spiked plasmid. (▲: Day 18–2.5 mg L⁻¹) and (●: Day 28–50 mg L⁻¹) represent the samples from the reactor test that were also sent to analyze for Hi-C sequencing. b Microbiome analysis from the samples sent for Hi-C sequencing at the genus level. c UpSet plot showing microbiome intersections between day 18 and day 28 from the test reactor

Fig. 5

Relative abundance of GFP and kanamycin resistance genes detected from the plasmid pBAV1K-T5-GFP relative to 16S rRNA gene in every sampling point. Only data from the reactor with free-floating plasmid displayed as non-detected values were detected for reactor control. Kanamycin concentrations are displayed on colored-background sections: 0.01, 2.5, 50, 100 mg L⁻¹. The plasmid was not spiked anymore in the test reactor (after day 36)

Fig. 6

Detection of microorganisms transformed by the synthetic plasmid using Hi-C sequencing. a Schematic representation of the discordant read analysis performed on contigs interacting with plasmid contigs, where synthetic constructs were generated to quantify the number of interactions from the Hi-C library alignment. b, c Taxonomic assignment of contigs that were linked to contigs harboring the information of the spiked pBAV1K-T5-GFP plasmid by frequency and by a normalized number of Hi-C links (#events divided by the length of the interacting contig). d Plasmid map containing the contigs used for the discordant read analysis. The kanR information in a, b is a combination of the number of interactions between the Kanamycin Resistance Gene with the NODE_74244 and NODE_147318 (I). Likewise, the GFP information is a combination of the number of interactions between the GFP gene with the NODE_147318 (II) and NODE_4094

Fig. 7

Detection of plasmids carrying aminoglycoside resistance in the microbial community by Hi-C sequencing. Comparison of the number of Hi-C interactions normalized by contig length (kb) between aminoglycoside resistance genes and bacterial hosts under exposures to kanamycin concentrations of 2.5 (a) and 50 (b) mg L⁻¹ in the test chemostat. Contigs containing aminoglycoside resistance genes were classified to determine their origin: genomic or plasmid DNA. Colors represent the number of Hi-C interactions normalized by the contig length (kb)