Mobile Gene Cassettes: A Fundamental Resource for Bacterial Evolution

Abstract

Horizontal gene transfer increases genetic diversity in prokaryotes to a degree not allowed by the limitations of reproduction by binary fission. The integron/gene cassette system is one of the most recently characterized examples of a system that facilitates horizontal gene transfer. This system, discovered in the context of multidrug resistance, is recognized in a clinical context for its role in allowing pathogens to adapt to the widespread use of antibiotics. Recent studies suggest that gene cassettes are common and encode functions relevant to many adaptive traits. To estimate the diversity of mobile cassettes in a natural environment, a molecular technique was developed to provide representative distributions of cassette populations at points within a sampling area. Subsequently, statistical methods analogous to those used for calculating species diversity were employed to assess the diversity of gene cassettes within the sample area in addition to gaining an estimate of cassette pool size. Results indicated that the number of cassettes within a 5x10-m sample area was large and that the overall mobile cassette metagenome was likely to be orders of magnitude larger again. Accordingly, gene cassettes appear to be capable of mobilizing a significant genetic resource and consequently have a substantial impact on bacterial adaptability.

Influence of industrial contamination on mobile genetic elements: class 1 integron abundance and gene cassette structure in aquatic bacterial communities

“…This is in large part because the primers used do not recover all integron-associated gene cassettes (Michael et al, 2004). Other potential biases relate to the use of gene cassette size as a proxy for distinguishing between different cassettes.…”

“…Other potential biases relate to the use of gene cassette size as a proxy for distinguishing between different cassettes. Previously, Michael et al (2004) detailed this potential bias, where the same cassette size class can contain multiple cassettes. However, this problem is not unique to this method, as the same limitation is present in techniques such as tRFLP and DGGE.…”

“…To assess the structure of the gene cassette community, fluorescently labeled primers targeting the attC site (HS286 and HS287 in Figure 1) were used to amplify gene cassettes from freshwater and estuarine sediment samples and microcosms as in Michael et al (2004). PCRs were run in triplicate for each sample consisting of 400 nM of each primer, 10 Â PCR buffer, 1.5 mM MgCl 2 , 200 mM dNTPs, 0.02 U JumpStart Taq (Sigma-Aldrich, St Louis, MD, USA) and 5 ng of template DNA in a 25-ml reaction volume.…”

“…PCR amplicons were subsequently analyzed using fragment analysis on an ABI 3130xl sequencer to separate gene cassettes by size, combining 1 ml of PCR product with 1.5 ml internal NAUROX size standard (to 1002 bp) (DeWoody et al, 2004) and 7.5 ml of HiDi/formamide. We limited analysis of cassette sizes to those within 250-999 bp, as the false-positive rate for PCR fragments smaller than 250 bp increases significantly (as determined from our sequencing results and as in Michael et al, 2004), and size class calling accuracy decreases with increasing amplicon size. To account for variability among PCR experiments, three triplicate PCRs were analyzed separately for each sample using GeneMapper (Applied Biosystems, Foster City, CA, USA) with the peak detection algorithm set to detect peaks greater than 20 fluorescence units above the threshold.…”

“…Class 1 integrons were first identified due to their role in the dissemination of antibiotic resistance gene cassettes in clinical bacteria (Stokes and Hall, 1989), but have since been found in a phylogenetically broad range of clinical and environmental bacteria including Gram-negative and Gram-positive bacteria (Nandi et al, 2004;Stokes et al, 2006;Boucher et al, 2007). Environmental surveys of integron-associated gene cassettes indicate that the majority of cassettes include novel sequences unrelated to known antibiotic resistance genes (Stokes et al, 2001;Holmes et al, 2003), with one conservative estimate of cassette richness being at least 2343 different gene cassettes in a 50-m 2 sediment plot (Michael et al, 2004). Integrons therefore represent a potential genetic resource by which bacteria in a variety of environments can adapt through the acquisition of new genetic material.…”