Worldwide prevalence of class 2 integrases outside the clinical setting is associated with human impact

Abstract

An intI-targeted PCR assay was optimized to evaluate the frequency of partial class 2-like integrases relative to putative, environmental IntI elements in clone libraries generated from 17 samples that included various terrestrial, marine, and deep-sea habitats with different exposures to human influence. We identified 169 unique IntI phylotypes (< or =98% amino acid identity) relative to themselves and with respect to those previously described. Among these, six variants showed an undescribed, extended, IntI-specific additional domain. A connection between human influence and the dominance of IntI-2-like variants was also observed. IntI phylotypes 80 to 99% identical to class 2 integrases comprised approximately 70 to 100% (n = 65 to 87) of the IntI elements detected in samples with a high input of fecal waste, whereas IntI2-like sequences were undetected in undisturbed settings and poorly represented (1 to 10%; n = 40 to 79) in environments with moderate or no recent fecal or anthropogenic impact. Eleven partial IntI2-like sequences lacking the signature ochre 179 codon were found among samples of biosolids and agricultural soil supplemented with swine manure, indicating a wider distribution of potentially functional IntI2 variants than previously reported. To evaluate IntI2 distribution patterns beyond the usual hosts, namely, the Enterobacteriaceae, we coupled PCR assays targeted at intI and 16S rRNA loci to G+C fractionation of total DNA extracted from manured cropland. IntI2-like sequences and 16S rRNA phylotypes related to Firmicutes (Clostridium and Bacillus) and Bacteroidetes (Chitinophaga and Sphingobacterium) dominated a low-G+C fraction ( approximately 40 to 45%), suggesting that these groups could be important IntI2 hosts in manured soil. Moreover, G+G fractionation uncovered an additional set of 36 novel IntI phylotypes (< or =98% amino acid identity) undetected in bulk DNA and revealed the prevalence of potentially functional IntI2 variants in the low-G+C fraction.

FIG. 1.

(a) Alignment of partial amino acid sequences of environmental integrases representative of each sampled site against functional integrase classes (1, 3, and 4) and tyrosine recombinases XerC and XerD. Numbering is based on the VchIntI4 sequence (38). The position of conserved motifs among integron-encoded integrases is indicated by segments labeled as Boxes I and II and Patches II and III and as additional domains (AD). The degree of conservation (percent identity) among equivalent residues is indicated as follows: black (100%), dark gray (95%), and light gray (85%). Conserved amino acids (93 to 99% identical) with functions related to protein folding, DNA binding, and recombination activity are highlighted in orange, pink (44), and red (38), respectively. Yellow columns show conserved proline residues (≥85% identical) detected within the AD region and conserved hydrophobic amino acids preceding functional residues exclusive of the VchIntI sequence. Green columns correspond to H, R, and Y residues from the conserved RHRY tetrad, which is characteristic of the entire tyrosine recombinase family (52). Numbers in boldface indicate the position of insertions and substitutions in functional integrases. GenBank accession numbers for the protein sequences of reference integrases (classes 1 to 4) and tyrosine recombinases XerC and XerD are AAQ16665, AAT72891, AAO32355, 99031763, P0A8P6, and P0A8P8, respectively. (b) Partial sequence alignment showing conserved residues (Q145 and W157; red columns) that seem to be exclusive of the Vibrio IntI clade.

FIG. 2.

Consensus neighbor-joining tree (compared positions for 108 amino acid residues), illustrating the relationship of representative IntI sequences recovered from a variety of marine and terrestrial habitats. Values represent the percentage of 1,000 bootstrap replications that supported the branching order. Roman numbers (I to III) designate clusters containing “clinical” integrases (classes 1 to 3). Red branches point out the arrangement of IntI sequences detected in environments with high fecal impact, while thicker black branches highlight the position of outgroup sequences (XerC and XerD recombinases) and reference sequences (best database matches). Numbers 1 to 7 denote clusters dominated by at least three soil IntI phylotypes originating from the same sample, whereas letters (A to G) indicate the location of clusters dominated by IntI phylotypes recovered from marine and deep-sea sediments (blue branches). The percent identities of IntI sequences associated with the highlighted clusters are shown in parentheses. Values of bootstrap support below 30% are not shown. GenBank accession numbers follow the designation of the reference and outgroup sequences.

FIG. 3.

Relative frequency distribution of 16S rRNA (a) and IntI phylotypes (b) in DNA fractions corresponding to 40 to 45% and 60 to 65% G+C contents, recovered from an agricultural soil from Michigan (AMS25) sampled after 1 month of manure application.