A comprehensive survey of integron-associated genes present in metagenomes

Abstract

Background: Integrons are genomic elements that mediate horizontal gene transfer by inserting and removing genetic material using site-specific recombination. Integrons are commonly found in bacterial genomes, where they maintain a large and diverse set of genes that plays an important role in adaptation and evolution. Previous studies have started to characterize the wide range of biological functions present in integrons. However, the efforts have so far mainly been limited to genomes from cultivable bacteria and amplicons generated by PCR, thus targeting only a small part of the total integron diversity. Metagenomic data, generated by direct sequencing of environmental and clinical samples, provides a more holistic and unbiased analysis of integron-associated genes. However, the fragmented nature of metagenomic data has previously made such analysis highly challenging.

Results: Here, we present a systematic survey of integron-associated genes in metagenomic data. The analysis was based on a newly developed computational method where integron-associated genes were identified by detecting their associated recombination sites. By processing contiguous sequences assembled from more than 10 terabases of metagenomic data, we were able to identify 13,397 unique integron-associated genes. Metagenomes from marine microbial communities had the highest occurrence of integron-associated genes with levels more than 100-fold higher than in the human microbiome. The identified genes had a large functional diversity spanning over several functional classes. Genes associated with defense mechanisms and mobility facilitators were most overrepresented and more than five times as common in integrons compared to other bacterial genes. As many as two thirds of the genes were found to encode proteins of unknown function. Less than 1% of the genes were associated with antibiotic resistance, of which several were novel, previously undescribed, resistance gene variants.

Conclusions: Our results highlight the large functional diversity maintained by integrons present in unculturable bacteria and significantly expands the number of described integron-associated genes.

Keywords: Antibiotic resistance; Functional annotation; Gene cassettes; Horizontal gene transfer; Integrons; Metagenomics; ORFans.

Fig. 1

Description of the computational pipeline used to detect attC sites in metagenomic data. Assembled metagenomic DNA sequences are used as input. Next, the gHMM-based HattCI is used to detect the attC sites present in the input sequences. Subsequently, the secondary structure of the detected attC sites is evaluated by a covariance model implemented in Infernal, which runs the search in its most sensitive mode. Identified attC sites on the same strand are considered to be part of the same integron when they are at maximum 4,000 nucleotides (nt) apart. Note that integrons with only one attC site are removed from the analysis in order to ensure a high true positive rate. Finally, the ORFs are predicted upstream of the attC sites

Fig. 2

Boxplots for a ORF length and b G/C-content for the integron-associated genes identified in this study. For comparisons, the corresponding data for three reference bacterial species have been included, Escherichia coli K-12, Staphylococcus aureus NCTC8325 and Bifidobacterium longum NCC2705. c Cluster analysis of the integron-associated genes. The x-axis shows the cluster threshold in sequence identity (higher value corresponds to a more homogeneous clusters) and the y-axis the number of produced clusters

Fig. 3

Functional annotation of the integron-associated genes (solid bars) and other genes found in metagenomes using COG functional categories (striped bars). Of the 13,397 integron-associated genes in our catalog, 2,277 genes matched a COG with a known function. 116,259,264 ORFs were not associated with integrons in metagenomes, out of which 50,201,496 matched a COG with a known function. Percentages on the plot are given in relation to those numbers

Fig. 4

Gene ontology analysis of the integron-associated genes using PFAM families. Out of the 13,397 integron-associated genes in our catalog, 3,488 matched a PFAM family with a known function, which were in turn mapped to the metagenomics GO slim. Not all PFAM families mapped to a GO term; as a result, 1534 genes had a corresponding GO term. Level 1 terms were removed and those with at least 5 counts were kept (For the whole list GO terms and their counts please see Additional file 3: Table S2)