Analysis by Mutagenesis of a Chromosomal Integron Integrase from Shewanella amazonensis SB2BT

Integrons are mobile genetic elements that can integrate and disseminate genes as cassettes by a site-specific recombination mechanism. Integrons contain an integrase gene (intI) that carries out recombination by interacting with two different target sites; the attI site in cis with the integrase and the palindromic attC site of a cassette. The plasmid-specified IntI1 excises a greater variety of cassettes (principally antibiotic resistance genes), and has greater activity, than chromosomal integrases. The aim of this study was to analyze the capacity of the chromosomal integron integrase SamIntIA of the environmental bacterium Shewanella amazonensis SB2BT to excise various cassettes and to compare the properties of the wild type with those of mutants that substitute consensus residues of active integron integrases. We show that the SamIntIA integrase is very weakly active in the excision of various cassettes but that the V206R, V206K, and V206H substitutions increase its efficiency for the excision of cassettes. Our results also suggest that the cysteine residue in the β-5 strand is essential to the activity of Shewanella-type integrases, while the cysteine in the β-4 strand is less important for the excision activity.Integrons are genetic elements that capture and rearrange genes that are contained within mobile gene cassettes by a mechanism of site-specific recombination mediated by an integrase (3). Several types of integron integrases have been described for clinical and environmental bacteria; classes 1, 2, and 3 integron integrases (1, 10, 11) and VchIntIA (17) and IntI9 (12) integrases are the only ones that are associated with antibiotic resistance genes. Some of these integrases were found exclusively on plasmids (IntI2*) (11) or on chromosomes (VchIntIA) (17), while others were found in both genetic contexts (IntI1) (7, 8, 20, 21). The efficiency of integron integrases to carry out cassette excision varies from one integrase to another and also depends on the structure and sequence of the attC sites located at both ends of the gene. IntI1 is generally the most active integrase, followed by IntI3. IntI2*179E and SonIntIA are less active but appear to tolerate more variation in attC sites. These enzymes could serve as models for determining important residues responsible for high levels of activity, using mutagenesis to substitute consensus residues and assaying for gain of function.Class 1 integrons, carrying the intI1 integrase gene, are generally associated with mobile elements, such as plasmids and Tn21-like transposons, and are most frequently found in clinical isolates (18). They are found mainly among gram-negative bacteria and especially among enterobacteria and pseudomonads (14). Class 1 integrons have also been found in some gram-positive bacteria, such as Enterococcus, Staphylococcus, and Corynebacterium (6). The clinical-type class 1 integrons (7) consist of two conserved regions and a variable region in which resistance genes are inserted in the form of cassettes (Fig. ​(Fig.1A).1A). These integrons were clearly derived from a structure related to Tn402, as they share many characteristics associated with this type of transposon (21). The common ancestor of clinical-type class 1 integrons was possibly a member of an integron pool that was acquired by diverse Betaproteobacteria (7). This hypothesis is based on the recent isolation of several new class 1 integron integrases from environmental DNA samples which are not associated with antibiotic resistance genes or with Tn402-like transposons (7, 8, 21).Open in a separate windowFIG. 1.(A) General structure of clinical-type class 1 integrons. Cassettes are inserted in the variable region of integrons by a site-specific recombinational mechanism. The attI1 and attC sites are shown by tiling and diagonal black lines, respectively, and promoters are denoted by P1, P2, P3, and P. Genes are as follows: intI1, integrase gene; qacEΔ1, antiseptic resistance gene; sul1, sulfonamide resistance gene; orf5, gene of unknown function. (B) Representation of the chromosomal integron of S. amazonensis SB2BT. The attI_Sam and attC sites are shown by a black box and horizontal black lines, respectively. Genes are as follows: SamintIA, integrase gene; orf, open reading frame gene.Class 2 integrons, carrying the intI2* integrase pseudogene, are present on Tn7 transposons and their derivatives (11). The intI2* gene encodes an integrase identical to 46% with IntI1, but its reading frame was interrupted by an early termination codon. The activity of this protein is restored when the stop codon at position 179 is replaced by a glutamate codon (11). Recently, two new intI2 genes were identified within integrons found in Providencia stuartii (2) and Escherichia coli (16). The sequences of these genes are not interrupted; position 179 is occupied by a glutamine codon, and the genes apparently code for functional enzymes. These intI2 genes each differ from intI2* of Tn7 at five positions (2, 16).Class 3 integrons, characterized by the presence of the intI3 gene, have been found in Serratia marcescens AK9373, in Klebsiella pneumoniae FFUL 22K isolated in Portugal, in four strains of Pseudomonas putida isolated in Japan, and more recently, in Delftia acidovorans C17 and Delftia tsuruhatensis A90 (1, 4, 19, 23). The IntI3 integrase has 61% identity with IntI1.The class 4 integron, with VchintIA, is an integron carried by the small chromosome of Vibrio cholerae O:1 569B (17). This integron contains more than 216 open reading frames (ORFs) coding for proteins of unknown functions associated with V. cholerae repetitive DNA sequence (VCR) elements to form 179 cassettes, and occupies about 3% of the bacterial genome.In recent years, the draft genomes of various environmental strains led to the identification of more than 100 new integron integrases. Among these, the SonintIA and NeuintIA integrase genes have been found, respectively, in genomes of Shewanella oneidensis MR-1 and Nitrosomonas europaea and shown to be active in cassette excision and integration (5, 13). Shewanella amazonensis SB2BT is an environmental gram-negative gammaproteobacterium that plays an important role in the bioremediation of contaminated metals and radioactive wastes (22). The U.S. Department of Energy Joint Genome Institute sequenced its 4.3-Mbp genome (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"CP000507","term_id":"119765642","term_text":"CP000507"}}CP000507). The genome encodes an integron integrase, SamIntIA, which is 64.8% identical to SonIntIA and 60.2% identical to IntI2* but only 46.9% identical to VchIntIA and 44.6% to IntI1. A sequence alignment of SamIntIA, SonIntIA, and IntI2* indicates that they are closely related, especially in the N-terminal and the C-terminal regions.Several residues of SamIntIA differed from a consensus alignment of active integron integrases. We wished to determine whether SamIntIA is active, compare its activity to that of SonIntIA and of IntI2*179E, and determine whether the alteration of certain residues affects its excision activity.