Novel Tn4371-ICE like element in Ralstonia pickettii and genome mining for comparative elements

Abstract

Background: Integrative Conjugative Elements (ICEs) are important factors in the plasticity of microbial genomes. An element related to the ICE Tn4371 was discovered during a bioinformatic search of the Ralstonia pickettii 12J genome. This element was analysed and further searches carried out for additional elements.A PCR method was designed to detect and characterise new elements of this type based on this scaffold and a culture collection of fifty-eight Ralstonia pickettii and Ralstonia insidiosa strains were analysed for the presence of the element.

Results: Comparative sequence analysis of bacterial genomes has revealed the presence of a number of uncharacterised Tn4371-like ICEs in the genomes of several beta and gamma- Proteobacteria. These elements vary in size, GC content, putative function and have a mosaic-like structure of plasmid- and phage-like sequences which is typical of Tn4371-like ICEs. These elements were found after a through search of the GenBank database. The elements, which are found in Ralstonia, Delftia, Acidovorax, Bordetella, Comamonas, Acidovorax, Congregibacter, Shewanella, Pseudomonas Stenotrophomonas, Thioalkalivibrio sp. HL-EbGR7, Polaromonas, Burkholderia and Diaphorobacter sp. share a common scaffold. A PCR method was designed (based on the Tn4371- like element detected in the Ralstonia pickettii 12J genome) to detect and characterise new elements of this type.

Conclusion: All elements found in this study possess a common scaffold of core genes but contain different accessory genes. A new uniform nomenclature is suggested for ICEs of the Tn4371 family. Two novel Tn4371-like ICE were discovered and characterised, using the novel PCR method described in two different isolates of Ralstonia pickettii from laboratory purified water.

Figure 1

Common core scaffold of Tn4371-like ICEs (in blue) and above inserted genes present in R. pickettii ICE_Tn43716033 (in yellow).

Figure 2

Use of the Artemis comparison tool to analysis Tn4371-like ICE sequences of Tn4371, R. pickettii 12J, both elements from D. acidovorans SPH-1 and C. testosteroni KF-1. All ICEs analysed shared extensive sequence homology, and general gene order. Arrows on top delimit the functional regions whose order is well conserved in all Tn4371-like ICEs.

Figure 3

Use of the Artemis comparison tool to analysis Tn4371-like ICE sequences of Tn4371, P. aeruginosa 2192, P. aeruginosa PA7, P. aeruginosa UCBPP-PA14 and P. aeruginosa PACS171b. All ICEs analysed shared extensive sequence homology, and general gene order. Arrows on top delimit the functional regions whose order is well conserved in all Tn4371-like ICEs.

Figure 4

Use of the Artemis comparison tool to analysis Tn4371-like ICE sequences of Tn4371, Shewanella sp. ANA-3, C. litoralis KT71, S. maltophilia K279a and Thioalkalivibrio sp. HL-EbGR7. All ICEs analysed shared extensive sequence homology, and general gene order. Arrows on top delimit the functional regions whose order is well conserved in all Tn4371-like ICEs.

Figure 5

Use of the Artemis comparison tool to analysis Tn4371-like ICE sequences of Tn4371, A. avenae subsp. citrulli AAC00-1, Acidovorax sp. JS42, B. petrii DSM12804, Diaphorobacter sp. TPSY and P. naphthalenivorans CJ2 plasmid pPNAP01. All ICEs analysed shared extensive sequence homology, and general gene order. Arrows on top delimit the functional regions whose order is well conserved in all Tn4371-like ICEs.

Figure 6

Amplification of genes of the putative Tn4371-like ICE ICE_Tn43716043 in Ralstonia pickettii strain ULM001 (a laboratory purified water isolate). A scheme of the amplified genes is shown above the 0.7% agarose gel of the PCR products generated with the primers listed in Table 2. Open white arrows denote ORFs of the Ralstonia pickettii ICE, and small black arrows represent the relative location of primers. Lanes M1 and M2 contain 200-10000 bp molecular size markers (Bioline Hyperladder I), respectively. The lanes and the product sizes are as follows: Lane 1, int gene and flanking bases (1035 bp); Lane 2 RepA gene (1657 bp), Lane 3 traG gene (1483 bp); Lane 4 trbI gene (1597 bp).

Figure 7

A) Schematic representation of Tn4371 excision and insertion into the R. pickettii chromosome. Primer LE1 and RE1 are the primers for detection of the circular form of the element. B) Agarose gel of attP of ICE_Tn43716043 and ICE_Tn43716044. Lanes M contains 200-10000 bp molecular size markers (Bioline Hyperladder I), Lane 1 ULM001, Lane2 ULM002, Lane 3 ULM006.