Integrase-Controlled Excision of Metal-Resistance Genomic Islands in Acinetobacter baumannii

Abstract

Genomic islands (GIs) are discrete gene clusters encoding for a variety of functions including antibiotic and heavy metal resistance, some of which are tightly associated to lineages of the core genome phylogenetic tree. We have investigated the functions of two distinct integrase genes in the mobilization of two metal resistant GIs, G08 and G62, of Acinetobacter baumannii. Real-time PCR demonstrated integrase-dependent GI excision, utilizing isopropyl β-d-1-thiogalactopyranoside IPTG-inducible integrase genes in plasmid-based mini-GIs in Escherichia coli. In A. baumannii, integrase-dependent excision of the original chromosomal GIs could be observed after mitomycin C induction. In both E. coli plasmids and A. baumannii chromosome, the rate of excision and circularization was found to be dependent on the expression level of the integrases. Susceptibility testing in A. baumannii strain ATCC 17978, A424, and their respective ΔG62 and ΔG08 mutants confirmed the contribution of the GI-encoded efflux transporters to heavy metal decreased susceptibility. In summary, the data evidenced the functionality of two integrases in the excision and circularization of the two Acinetobacter heavy-metal resistance GIs, G08 and G62, in E. coli, as well as when chromosomally located in their natural host. These recombination events occur at different frequencies resulting in genome plasticity and may participate in the spread of resistance determinants in A. baumannii.

Keywords: Acinetobacter baumannii; copper resistance; genomic island; integrase; mobile genetic element.

Figure 1

Schematic map of the Acinetobacter baumannii strain genomic islands G08 and G62 and phylogenetic tree of genomic island (GI) integrases. (A) G62 island is taken from strain ATCC 17978 (accession CP000521; updated refseq NC_009085.1) and G08 from both strains AB0057 (NC_011586.2/CP001182.2) and AYE (NC_010410.1/CU459141.1). The annotated sequences were aligned and visualized by the Easyfig tool [21]. The genes involved in encoding copper efflux systems are shown in blue, all czc-like genes czcA, czcB, and czcC encoding cadmium, zinc, and cobalt resistance are represented in green color, genes encoding ferrous iron transport proteins are shown in red (feoA and feoB), and a putative further heavy metal efflux system A1S_2929 is shown in pink. The integration sites (att sites) for both GIs are shown as black vertical lines. The genes encoding the integrases are colored in orange and all other genes in grey. Locus tags of relevant genes are shown above or below the genes. The image is drawn in scale and the percentage of DNA identity between various regions is shown by gradient shading. (B) Phylogenetic tree of tyrosine recombinases from strains ATCC 17978, AB0057, and AYE. The evolutionary relationship between phage integrase family proteins (NCBI Reference Sequence) detected in three strains of A. baumannii was inferred using the Neighbor-Joining method [22]. Integrases were labelled according to the genomic island number, strain name, followed by Refseq accession numbers. In certain instances, some GIs have not been given a specific number, but instead were indicated by their accession. When two strains are mentioned next to a GI, this means that the same GI is present in both strains. Identical proteins in different genomes which yield identical Refseq numbers are shown only once. The three major branches are indicated by numbers on the right side of the figure. G08 and G62 are indicated by black boxes.

Figure 2

Integrase distribution on an A. baumannii phylogenetic tree. The whole genome phylogenetic SNP tree was built using the FFP and input from the 105 complete A. baumannii genomes deposited in GenBank (08/07/2018) with the addition of strains A424 and KR3831. Strain names include the MLST “Pasteur” sequence type. The matrix of 16 integrases on the right part of the figure includes all integrase genes present in AB0057, AYE, and ATCC 17978 (red absent; blue present). The integrases of the GI are numbered according to Di Nocera et al. [14]. Three further integrases present in the three stains but not associated to genomic islands were included in the analysis and named int1 (ABAYE_RS10930), int2 (AUO97_RS03560), and the p3ABAYE integrase int3 (ABAYE_RS00155).

Figure 3

G08 and G62 mini-GI vectors for E. coli and A. baumannii. pUC18 (A) used as a vector harboring bla gene conferring ampicillin resistance (green), E. coli origin of replication ori (grey), multiple cloning site MCS shown as green triangle, to clone mini-GIs containing upstream and downstream flanking borders with attL/attR sites (red) of the respective islands as well as integrase genes (B) G08int (olive) and (C) G62int (blue). The IPTG-inducible Plac promoter was fused by PCR in front of the integrase genes during construction of the plasmids (B,C). Plasmids used to assess integrase activity in A. baumannii (D–F) were constructed by fusing pUC18-based G08 and G62 mini-island vectors with pWSK129 carrying the aphA1 conferring kanamycin resistance (orange) and pLG339 replication initiation protein (repP, light green). To allow for stable transfer of the constructs to Acinetobacter pWSK129-WH-G08 (G) and pWSK129-WH-G62 (H) were constructed. pWSK129-kan plasmids were used to clone the Acinetobacter origin of Replication (oriR) from pWH1266 (black color) resulting in pWSK129-WH-derived new constructs compatible with A. baumannii strains.

Figure 4

Integrase induction drives excision of G08 and G62 mini-islands in E. coli and A. baumannii. Gel images of circularized G08 (from AB0057) (A) and G62 (from ATCC 17978) (B) mini-islands respectively, with (+) and without (−) IPTG induction. Mini-GI excision was tested by amplification of circular intermediates using outwards facing primer sets yielding 567 bp product for G08 (A) and a 489 bp product for G62 excision (B). Different time points (in hours) after IPTG induction in early exponential phase are shown (legend above gel). Integrase dependent excision of G08 and G62 mini-islands in A. baumannii is shown in panel C. Gel images of circularized G08 and G62 mini-islands 8 h after IPTG induction with the same primers as in E. coli (panel A and B). The non-induced samples in lane 3 for G8 and in lane 5 for G62 in lanes 4 and 5 of panel C. The marker is Gene Ruler 1 kb and the size of some bands in bp is given on the left of each gel.

Figure 5

Allelic variation in the attB site of A. baumannii strains. (A) Shows the variability of att sequences in AB0057 and AYE and KR3831 compared to ATCC 17978. The att sites sequenced in this study are abbreviated as (seq). (B) Shows two variable alleles of attB sequences in dusA in two A. baumannii strains ATCC 17978 and AB307-0294 lacking the G08 island. The attB site is underlined. The variable alleles differing from the consensus sequence by two or three SNPs are shown in red.

Figure 6

Quantification of G08 and G62 island excision and integrase expression in A. baumannii strains with and without mitomycin C induction. The data in (A) represent the real-time PCR detection of reconstituted target sites after excision of G08 (excision) and of the circular intermediates (circularization) without any induction. The G08 positive strains are AB0057 strain (black, accession NC_011586.2/CP001182.2). AYE (white, accession NC_010410.1/CU459141.1), A424 (striped, accession GCA_003185755.1), KR3831 (grey, accession: GCF_003185745.1. GCA_003185745.1), and data are normalized to AB0057. (B) Reports the variation in G08 excision by detecting circular intermediates with and without mitomycin C induction (0.75 × MIC) for 2 h. The asterisks represent the significance of change in each strain when compared to AB0057. Error bars represent SEM of three independent replicates. (C) Repost real-time PCR quantification of G62 circular intermediates with and without mitomycin C induction. (D) Shows the expression G08int (A424_1287 from strain A424, protein ID: PRJNA473420:DMB35_05960, Genbank accession: GCA_003185755.1) and G62int (A1S-2927 from ATCC 17978) measured by real-time PCR without or with mitomycin C exposure (grey). Error bars indicate the SEM of three independent replicates in each experiment as analyzed by two-way ANOVA test. ** p < 0.01, *** p < 0.001.