The Integrative Conjugative Element clc (ICEclc) of Pseudomonas aeruginosa JB2

Abstract

Integrative conjugative elements (ICE) are a diverse group of chromosomally integrated, self-transmissible mobile genetic elements (MGE) that are active in shaping the functions of bacteria and bacterial communities. Each type of ICE carries a characteristic set of core genes encoding functions essential for maintenance and self-transmission, and cargo genes that endow on hosts phenotypes beneficial for niche adaptation. An important area to which ICE can contribute beneficial functions is the biodegradation of xenobiotic compounds. In the biodegradation realm, the best-characterized ICE is ICEclc, which carries cargo genes encoding for ortho-cleavage of chlorocatechols (clc genes) and aminophenol metabolism (amn genes). The element was originally identified in the 3-chlorobenzoate-degrader Pseudomonas knackmussii B13, and the closest relative is a nearly identical element in Burkholderia xenovorans LB400 (designated ICEclc-B13 and ICEclc-LB400, respectively). In the present report, genome sequencing of the o-chlorobenzoate degrader Pseudomonas aeruginosa JB2 was used to identify a new member of the ICEclc family, ICEclc-JB2. The cargo of ICEclc-JB2 differs from that of ICEclc-B13 and ICEclc-LB400 in consisting of a unique combination of genes that encode for the utilization of o-halobenzoates and o-hydroxybenzoate as growth substrates (ohb genes and hyb genes, respectively) and which are duplicated in a tandem repeat. Also, ICEclc-JB2 lacks an operon of regulatory genes (tciR-marR-mfsR) that is present in the other two ICEclc, and which controls excision from the host. Thus, the mechanisms regulating intracellular behavior of ICEclc-JB2 may differ from that of its close relatives. The entire tandem repeat in ICEclc-JB2 can excise independently from the element in a process apparently involving transposases/insertion sequence associated with the repeats. Excision of the repeats removes important niche adaptation genes from ICEclc-JB2, rendering it less beneficial to the host. However, the reduced version of ICEclc-JB2 could now acquire new genes that might be beneficial to a future host and, consequently, to the survival of ICEclc-JB2. Collectively, the present identification and characterization of ICEclc-JB2 provides insights into roles of MGE in bacterial niche adaptation and the evolution of catabolic pathways for biodegradation of xenobiotic compounds.

Keywords: ICEclc; PCBs; Pseudomonas aeruginosa; biodegradation; chlorobenzoates; integrative conjugative element (ICE); xenobiotic metabolism.

FIGURE 1

Overview of ICEclc-JB2 structure (A) and comparison to ICEclc-LB400 (B) and ICEclc-B13 (C). In each panel, the element length is indicated by the numbered line and regions carrying cargo genes and core genes are shaded blue and beige, respectively. Graphs below each element display the %G + C skew. For ICEclc-JB2, green regions are conserved with both ICEclc-LB400 and ICEclc-B13, yellow regions are shared with ICEclc-LB400 only and blue regions are not shared with either ICEclc-LB400 or ICEclc-B13. The numbers across the top of these regions in ICEclc-JB2 are used to identify their locations in ICEclc-LB400 and ICEclc-B13. In ICEclc-JB2, the red triangle between regions 5 and 6 indicates the location of the amp cluster that is present in ICEclc-LB400 and ICEclc-B13 (red region, B,C), but absent in ICEclc-JB2. The purple triangles between regions 3 and 4, and between regions 6 and 7 indicate the locations of sequence occurring only in ICEclc-LB400 (purple bars labeled “a” and “b”; B). For ICEclc-B13 (C) the multicolor triangle indicates the location at which regions occur in ICEclc-JB2 and/or ICEclc-LB400, but are absent from ICEclc-B13. The purple triangle above ICEclc-B13 regions 6 and 7 indicates the location of sequence occurring only in ICEclc-LB400 (purple bar labeled “b”; B). The sizes of the elements presented are: 123,270 bp (ICEclc-JB2), 122,836 bp (ICEclc-LB400) and 105,032 bp (ICEclc-B13).

FIGURE 2

Repeat regions in the ICEclc-JB2 cargo area. (A) Overview of the location and orientation of the repeat regions within ICEclc-JB2 cargo area. The color-coding and numbering of bars is as described in Figure 1. The locations of transposases/insertion sequence elements are indicated by gold circles. (B) Detail of the repeat regions showing gene structure, numbering across the top of each repeat is the base pair location in the P. aeruginosa JB2 genome. Genes are numbered 1–20 based on structure in Repeat 2. Color-coding of genes is as follows: predicted transposase (gold; gene 1), ohbBAR (light blue; genes 2–4, respectively), predicted catabolic enzymes (green; genes 5, 7, 8), hybIHGEF (orange, genes 9-13, respectively), hybDCABR (purple; genes 14, 15, 17–19 respectively), hypothetical (black; genes 6, 16, 20). In Repeat 1, frame shifts result in the absence of genes 5 and 9 and the replacement of genes 7, 11, and 13 with fragmented ORFs (indicated by “X”). In (B), Repeat 2 spans (left to right) genome positions 4,949,315–4,933,315 and Repeat 1 spans genome positions 4,971,315–4,955,315 (left to right).

FIGURE 3

PCR Analysis of ICEclc-JB2 structure in wild type P. aeruginosa JB2 and a spontaneous mutant of strain JB2 deficient in growth on 2-CBa. (A) Map of ICEclc-JB2 displaying locations of 11 PCR primer pairs used to probe its structure. The gray box indicates expanded detail of the repeat region. Color-coding of genes follows is as used in Figure 2. The symbols “+” or “–“ beneath numbers 1–11 indicate the presence or absence, respectively of a PCR product in the mutant for the indicated primer pair. For primer pair 2, the symbol “(+)” is placed beneath its location between repeats 1 and 2 to indicate that the product observed in the mutant was likely derived from one or both of its other two binding sites that are located at the termini of the repeats. (B) Agarose gel of PCR products from the wild type. (C) Agarose gel of PCR products from the mutant. Numbering across the top of each gel corresponds to the primer pair location indicated in (A). Numbering along the left side of gels indicates the size of the five bottom bands in the DNA ladder.