Natural transformation with synthetic gene cassettes: new tools for integron research and biotechnology

Abstract

Integrons are genetic elements that can capture and express genes packaged as gene cassettes. Here we report new methods that allow integrons to be studied and manipulated in their native bacterial hosts. Synthetic gene cassettes encoding gentamicin resistance (aadB) and green fluorescence (gfp), or lactose metabolism (lacZY), were made by PCR and self-ligation, converted to large tandem arrays by multiple displacement amplification, and introduced into Escherichia coli or Pseudomonas stutzeri strains via electroporation or natural transformation. Recombinants (Gm R or Lac + ) were obtained at frequencies ranging from 10 1 to 10 6 c.f.u. (mg DNA) "1 .Cassettes were integrated by site-specific recombination at the integron attI site in nearly all cases examined (370/384), including both promoterless and promoter-containing cassettes. Fluorometric analysis of gfp-containing recombinants revealed that expression levels from the integron-associated promoter P C were five-to 10-fold higher in the plasmid-borne integron In3 compared with the P. stutzeri chromosomal integrons. Integration of lacZY cassettes into P. stutzeri integrons allowed the bacteria to grow on lactose, and the lacZY gene cassette was stably maintained in the absence of selection. This study is believed to be the first to show natural transformation by gene cassettes, and integron-mediated capture of catabolic gene cassettes. INTRODUCTIONMultiple antibiotic resistance in bacteria is a problem for human health (Davies & Davies, 2010), and is associated with the actions of mobile genetic elements (MGEs) such as plasmids, transposons and integrons (Frost et al., 2005). Integrons can assemble resistance genes from diverse sources at a single genetic locus (Hall & Collis, 1998;Mazel, 2006; Schlüter et al., 2007;Stokes & Hall, 1989; Gillings et al., 2008). The integron consists of the intI gene encoding integrase, an attachment site (attI), a promoter (P c ), and an array of mobile gene cassettes (Nunes-Düby et al., 1998;Recchia & Hall, 1997;Stokes & Hall, 1989; Lévesque et al., 1994;Jové et al., 2010). The cassettes typically consist of a single promoterless ORF and a recombination site (attC). The integron integrase recombines attC with attI, or attC with attC, enabling both integration and excision of gene cassettes, which shift between free circular and linear integrated forms. Integrons are common in bacteria, and diverse in their phylogeny and predicted functions (Boucher et al., 2007a; Elsaied et al., 2007;Gillings et al., 2005;Holmes et al., 2003a;Nield et al., 2001;Rowe-Magnus & Mazel, 2001;Rowe-Magnus et al., 2003). Integrons increasingly seem to have a role as general-purpose agents of adaptation, not just enablers of antibiotic resistance (Koenig et al., 2009(Koenig et al., , 2011Rosewarne et al., 2010;Wright et al., 2008).Previous studies have used excision assays (Collis & Hall, 1992;Drouin et al., 2002; Léon & Roy, 2003) or cointegrate assays (Biskri et al., 2005;Collis et al., 2002Collis et al., , 2001Holmes et al., 2003b;Martinez &...

Lateral Antimicrobial Resistance Genetic Transfer is active in the open environment

“…Unexpectedly, this assay showed a high frequency (46%) and diversity of genera with natural competence for the insertion of wild-type ARGCs, which were against the prediction of the most accepted hypothesis. In a previous study, native chromosomal integrons from P. stutzeri strains 17587 and 17641 have been shown to acquire synthetic gene cassettes 39 and environmental A . baylyi ADP1 has been shown to acquire complete genetic platforms of class 1 integrons 38 , both via natural transformation.…”

“…This methodological approach differed from previous similar studies which were performed with intI1 “clinical” alleles 23 under antibiotic pressure, usually with overexpressed integrases in E . coli 30–39 . Eleven environmental strains belonging to 8 genera of γ- Proteobacteria harbouring the complete intI1 gene, isolated from different substrates, including soil, river water and fox faeces, were selected for recombination assays (Supplementary Table S1, Fig.…”

Integrons: Past, Present, and Future

“…Synthetic and natural gene cassettes can readily be introduced into cells via natural transformation. This potentially allows any gene to be incorporated into an integron (247). Further, marker cassettes, such as gene cassettes encoding green fluorescent protein, could be used to naturally transform environmental bacteria with active integron recombination systems.…”

The Integron: Adaptation On Demand

“…An integron-based cloning technique has also been developed, based on the delivery of synthetic cassettes into the genetic backbone of both MIs and CIs via natural transformation (129). This technique can be used with wild-type environmental bacteria, does not require a vector, can yield high frequencies of recombinants in favorable conditions, and recombinants are stable in the absence of selection.…”