Dissecting molecular evolution of class 1 integron gene cassettes and identifying their bacterial hosts in suburban creeks via epicPCR

Objectives
Our study aimed to sequence class 1 integrons in uncultured environmental bacterial cells in freshwater from suburban creeks and uncover the taxonomy of their bacterial hosts. We also aimed to characterize integron gene cassettes with altered DNA sequences relative to those from databases or literature and identify key signatures of their molecular evolution.
Methods

We applied a single-cell fusion PCR-based technique—emulsion, paired isolation and concatenation PCR (epicPCR)—to link class 1 integron gene cassette arrays to the phylogenetic markers of their bacterial hosts. The levels of streptomycin resistance conferred by the WT and altered aadA5 and aadA11 gene cassettes that encode aminoglycoside (3″) adenylyltransferases were experimentally quantified in an Escherichia coli host.
Results

Class 1 integron gene cassette arrays were detected in Alphaproteobacteria and Gammaproteobacteria hosts. A subset of three gene cassettes displayed signatures of molecular evolution, ..., Approximately 200 mL freshwater samples were collected from Kikkiya Creek (-33.776, 151.117) and the Macquarie University Lake section of Mars Creek (-33.772, 151.115) in three independent biological replicates in suburban metropolitan Sydney, NSW, Australia (Figure 1A). Water samples were immediately transported back to the lab, filtered with 40 µm EASYstrainer cell strainers (Greiner AG, Germany) and centrifuged at 3,220 g for 15 minutes at 4˚C. Pellets were resuspended in 300 µL 0.01M phosphate buffered saline (PBS) solution (pH 7.4) and stored as 25% glycerol stocks for cryopreservation. Estimates of bacterial cell density were obtained by fluorescence microscopy using previously described procedures (1).
epicPCR to determine the identity of bacterial hosts of class 1 integrons in suburban creeks was performed in three technical replicates for each of the three biological replicates using previously established procedures with minor modifications (1). Approximately 150,000 cells wer..., Users in the UNIX/Linux environment can use the unzip command to decompress the downloaded .ZIP file. All Nanopore sequence filtering and processing steps for epicPCR amplicon reads were carried out using an in-house pipeline (https://github.com/timghaly/Int1-epicPCR). First, the pipeline quality-filters Nanopore reads using NanoFilt v2.8.0 (1), removing those with an average read quality of less than 7 and read length less than 670 bp, which represents the minimum length of an epicPCR product with a cassette-less integron. Quality-filtered reads are then oriented and trimmed with the final nested epicPCR forward and reverse primer sequences using Pychopper v2.7.0 (https://github.com/epi2me-labs/pychopper). Pychopper identifies both primers in each read using edlib v1.2.3 (2), and orients the reads based on the forward and reverse primer sequences. Reads that do not contain both primers in the correct orientation are discarded. The pipeline then clusters the primer-oriented reads into a...,