The integrase of genomic island GI sul2 mediates the mobilization of GI sul2 and IS CR-related element CR2- sul2 unit through site-specific recombination

Abstract

In the worldwide health threat posed by antibiotic-resistant bacterial pathogens, mobile genetic elements (MGEs) play a critical role in favoring the dissemination of resistance genes. Among them, the genomic island GIsul2 and the ISCR-related element CR2-sul2 unit are believed to participate in this dissemination. However, the mobility of the two elements has not yet been demonstrated. Here, we found that the GIsul2 and CR2-sul2 units can excise from the host chromosomal attachment site (attB) in Shigella flexneri. Through establishing a two-plasmid mobilization system composed of a donor plasmid bearing the GIsul2 and a trap plasmid harboring the attB in recA-deficient Escherichia coli, we reveal that the integrase of GIsul2 can perform the excision and integration of GIsul2 and CR2-sul2 unit by site-specific recombination between att core sites. Furthermore, we demonstrate that the integrase and the att sites are required for mobility through knockout experiments. Our findings provide the first experimental characterization of the mobility of GIsul2 and CR2-sul2 units mediated by integrase. They also suggest a potential and unappreciated role of the GIsul2 integrase family in the dissemination of CR2-sul2 units carrying various resistance determinants in between.

Keywords: ISCR elements; genomic island (GI); integrase; mobilization; site-specific recombination.

Figure 1

Genetic organization of genomic island GIsul2 in S. flexneri 2a str. 2457T (accession AE014073) or S. flexneri 51575 (accession AZPD01000036, position 2108-17567). (A) Organization of the GIsul2 element. The thick central line represents the backbone of the GIsul2 with flags representing the three attachment sites (attL/attLR/attR). Horizontal arrows indicate the position, size, and orientation of ORFs with the names below. The guaA gene encoding a guanosine monophosphate synthetase (GMP) is shown in teal. The int gene encoding the integrase is represented in red. Bright green, light red, and pink ORFs denote alpA, stbC, and resG genes, respectively. Genes putatively involved in replication, transfer-related, toxin-antitoxin system, arsenic resistance, and the CR2-sul2 unit are shown in purple, blue, cyan, pink and green, respectively. Gray ORFs encode hypothetical proteins. (B) Nucleotide alignment of att sites of GIsul2. The dotted box represents the expected att sites recognized by the integrase. The orange underlined motif indicates the putative core recombination region.

Figure 2

Excision of GIsul2 and of CR2-sul2 unit from the chromosome of S. flexneri 51575. (A) The predicted circular intermediates resulting from recombination between the three att sites of GIsul2. The crossed arrow indicates that the event could not be observed. Solid flags denote the various att sites. (B) Nested PCR assays for the detection of the three potential circular intermediates: Mw, M5 DL2000 plus DNA Marker (Mei5 Biotechnology, Beijing); lane 1, CR2-sul2 circular intermediate; lane 2, GI12K circular intermediate; lane 3, GIsul2 circular intermediate. (C) Alignment of the sequenced junctions resulting from the circularization of GIsul2 (attP_GIsul2) and of CR2-sul2 unit (attP_CR2−sul2) with their corresponding initial att sites.

Figure 3

Co-integrate formation in E. coli DH5α through site-specific recombination at the att sites. (A) Construction of GItetWsul2. The gray shadings denote 100% identity between GItetWsul2 and the original GIsul2. ORFs (horizontal arrows) are colored as in Figure 1. (B) Detection of circular intermediates excised from the donor plasmid pKDGItetWsul2 in E. coli DH5α. Mw, DNA size marker; lane 1, CR2-sul2 unit circular intermediate; lane 2, GItetWsul2 circular intermediate. (C) Organization of the donor (pKDGItetWsul2) and trap (pFKattB) plasmids used in the two-plasmid integration experiment, and expected recombinant structures. Intervening ORFs are depicted with filled arrows while red, green, blue, and black flags denote attL, attLR, attR, and attB, respectively. The putative hybrid attachment sites resulting from site-specific recombination are represented by two-color flags. Primers used for the detection of the various structures are indicated. (D) Couples of primer pairs used to detect co-integrate structures in recombinant bacteria, and their expected amplification size. (E) PCR amplification using the six primer pairs from (D) on the plasmid extracts of six independent recombinant colonies. Colonies are labeled 1–6, with the six primer pairs amplification (A–E) displayed below each of them.

Figure 4

Detection of pKFattB-GItetWsul2 and pKFattB-CR2sul2 structures in recombinant colonies. PCR amplification using the primer pairs Sul2-F/Int-R and Sul2-F/CR2-R for the detection of GItetWsu2-pKFattB (A) and pKFattB-CR2sul2 (B) structures, respectively. PCR reactions were conducted on the same six recombinant colonies as in Figure 3E.

Figure 5

Integration of circular intermediates into the trap plasmid pKFattB under sucrose negative selection. (A) XbaI digestion of the plasmid extract from six independent recombinant colonies. Bands at 8,503 bp correspond to the recombinant plasmid pKFattB-CR2sul2 (CR2-sul2 inserted into pKFattB) and bands at 2,843 bp correspond to original pKFattB copies. (B) XbaI and BamHI double restriction of the same plasmid extracts. BamHI cuts CR2-sul2 unit once and not pKFattB, leading to the digestion of pKFattB-CR2sul2 into 6,500 and 2,003 bp bands. (C) Detection of GItetWsu2-pKFattB structure. Left panel, XbaI digestion of the plasmid extract from six independent recombinant colonies. M1, M2, different DNA markers. Right panel, XbaI and SacI double restriction of plasmid extracts (number 5 and 6). SacI cuts GItetWsu2 once and not pKFattB, leading to the digestion of GItetWsu2-pKFattB into 13,276 and 739 bp bands. The band at 2.8 kb is the residual pKFattB plasmid. The bottom schematics exhibit the locus of restriction enzymes and expected digestion sizes for restriction analysis.

Figure 6

The distribution of GIsul2, CR2-sul2 unit, and GI12K in chromosomes and plasmids of NCBI complete genome database. The internal species tree represent the host distribution of the three elements. The size of the hexagon and star markers denotes the number of genomes of each host carrying the corresponding elements in chromosomes or plasmids, respectively. The bar height in the inner circle represents the proportion of genomes carrying the elements among the total genome of the species in the database. The six outermost heatmaps represent each element's abundance, separated by chromosomal or plasmid localization. The color intensity corresponds to the proportion of the host in the total number of genomes carrying the given element. Orange, olive green, and purple heatmaps represent the distribution of GIsul2, GI12K, and the CR2-sul2 unit in chromosomes, respectively. Blue and green heatmaps represent the dissemination of GIsul2 and CR2-sul2 units in the plasmids, respectively. Hosts with a total number of genomes >10 are highlighted and abbreviated as follows: Kp, Klebsiella pneumoniae; Ec, Escherichia coli; Se, Salmonella enterica; Ab, Acinetobacter baumannii.