Co-assortment in integron-associated gene cassette assemblages in environmental DNA samples

Abstract

Background: It has been shown that integron-associated gene cassettes exist largely in tandem arrays of variable size, ranging from antibiotic resistance arrays of three to five cassettes up to arrays of more than 100 cassettes associated with the vibrios. Further, the ecology of the integron/gene cassette system has been investigated by showing that very many different cassettes are present in even small environmental samples. In this study, we seek to extend the ecological perspective on the integron/gene cassette system by investigating the way in which this diverse cassette metagenome is apportioned amongst prokaryote lineages in a natural environment.

Results: We used a combination of PCR-based techniques applied to environmental DNA samples and ecological analytical techniques to establish co-assortment within cassette populations, then establishing the relationship between this co-assortment and genomic structures. We then assessed the distribution of gene cassettes within the environment and found that the majority of gene cassettes existed in large co-assorting groups.

Conclusions: Our results suggested that the gene cassette diversity of a relatively pristine sampling environment was structured into co-assorting groups, predominantly containing large numbers of cassettes per group. These co-assorting groups consisted of different gene cassettes in stoichiometric relationship. Conservatively, we then attributed co-assorting cassettes to the gene cassette complements of single prokaryote lineages and by implication, to large integron-associated arrays. The prevalence of large arrays in the environment raises new questions about the assembly, maintenance and utility of large cassette arrays in prokaryote populations.

Figure 1

Non-metric Multidimensional Scaling (nMDS) plot of the Gene Cassette assemblage collected from five distances (0, 0.01, 0.1, 0.5, 1, 5 m labelled A-F inclusive) along the two transects (1 and 2) 10 metres apart.

Figure 2

A subset of a large co-assorting group (Groups A and B, additional file 1) seen in multiple DNA samples. Co-assorting groups share similar peak intensity distributions between environmental DNA samples. For example, the arrowed peaks representing 429 bp and 482 bp size classes retain their relative heights in all samples in which they are present.

Figure 3

Quantitation of potentially linked gene cassettes using QPCR. In this series of panels, the left hand vertical axis represents abundance as measured by QPCR. The horizontal axis indicates the different environmental DNA samples in which the abundance of the target gene cassettes was measured. The right hand vertical axis shows the ratio of the abundances of the two gene cassettes considered, demonstrating a stoichiometric relationship through a constant ratio between DNA samples. Graph A indicates the 429/482 bp co-assorting pair, Graph B indicates the 258/347 bp co-assorting pair and Graph C shows the 253/325 bp non co-assorting pair as discussed in the text.