Microbial community analysis
Genomic DNA was extracted using the Ultra Clean Soil DNA extraction kit
(QIAGEN, Hilden, Germany) following manufacturer’s instructions, with
the exception of heating the samples for 5 minutes at 65°C prior to bead
beating. DNA extracts were checked on a 1% agarose gel. High molecular
weight DNA was obtained (>10 kb) with a concentration of 10
ng µL-1 or higher. Extracted DNA was stored at -20°C
until further use.
Analysis of the V3-V4 region of the 16S rRNA gene was conducted using
amplicon sequencing. The extracted DNA was sent for amplification and
sequencing at a commercial company (Novogene, China). Amplification was
achieved using the universal primer set 341f (CCTAYGGGRBGCASCAG) / 806r
(GGACTACNNGGGTATCTAA T) (Muyzer et al., 1993)(Caporaso et al., 2011).
All polymerase chain reactions (PCR) were carried out in 30 µL reactions
with 15 µL of Phusion® High_fidelity PCR Master Mix (New England
Biolabs, USA), 0.2 µmol L-1 of forward and reverse
primers, and 10 ng template DNA. Thermal cycling started with
denaturation at 98°C for 10 s, annealing at 50°C for 30 s, and
elongation at 72°C for 60 s for 30 cycles, prior to ending with 72°C for
5 min. These pools of amplicons were sequenced using an Illumina
HiSeq2500 platform. The sequencing datasets were cleaned and trimmed
according to Jia et al. (2016) and processed with Qiime (Caporaso
et al., 2010) using Uparse with a 97% stringency to yield operational
taxonomic units (OTUs). OTUs were taxonomically classified using the
Mothur classifier (Wang et al., 2007) with 0.8 confidence interval
against the SILVA database 123 release of July 2015. The clean and
trimmed sequences can be retrieved at NCBI using accession number
SAMN11350619 - SAMN11350630. The inoculum was sequenced twice as a
technical replicate, this data can be retrieved at NCBI (data not
presented in this paper).
Cell fixation and fluorescence in situ hybridisation (FISH) were
carried out as described by Johnson et al. (Johnson et al., 2009)
using the probes listed in Table S2, except that hybridization was
carried out overnight. Additionally, DAPI staining was used to stain all
microbial cells by incubating the multi-wells microscopy slides of fixed
cells with 10 µL of a solution of 10 mg DAPI mL-1 per
well for 15 min. The samples were analysed using an epifluorescence
microscope, Axioplan 2, (Zeiss, Oberkochen, Germany). Digital images
were acquired using a Zeiss MRM camera together with Zeiss imaging
software AxioVision version 4.7.