The genomic island we identified in Pseudomonas sp. strain B13, the first bacterium described to degrade 3-chlorobenzoate, has been called the clc element (or ICEclc) because of its ability to provide chlorocatechol degradation to the host. The clc element has a size of 105 kb and we know of one natural variant which is very closely related. This clc like element was present in a Ralstonia sp. degrading chlorobenzenes and was isolated from contaminated groundwater in the United States. We and others showed that the clc element is self-transferable in laboratory 'matings' at high frequencies, but also transfers in more complex communities and technical systems from strain B13 to other Beta- and Gammaproteobacteria, such as Ralstonia, Pseudomonas putida, Burkholderia sp. and P. aeruginosa. The work on Ralstonia demonstrated that the clc element is not a laboratory artifact but present in the natural environment and can acquire gene fragments from other related species.ICEclc is completely capable to self-transfer from the strain B13 to an intermediate recipient and from there to another species. Details on the transfer process are still mostly lacking.
Depending on the host, the clc element can be present in one, two, or more copies. Also in strain B13 two copies are present. In all hosts examined, the clc element resides site-specifically in the chromosome at the most 3' 18-bp sequence of a tRNAGly gene. At the other end of the element, a copy of this 18-bp sequence is found. A critical feature of the clc element's life-style is formed by the integrase, the gene of which (intB13) is found close to the tRNAGly insertion site and oriented away from it. A cloned intB13 integrase in E. coli carries out the site-specific recA independent integration of a plasmid with the 18-bp repeat sequence into a second plasmid carrying the tRNAGly gene. This showed that the integration process into the chromosome is mediated by the IntB13 integrase enzyme. The reverse process, excision, has not been reproduced in E. coli, but small amounts of the excised clc element can be detected in cultures of strain B13 itself by Southern hybridization and PCR.
Sequencing of this excised form showed that it consists of a closed DNA molecule in which both 18-bp repeats have recombined again to one. The current hypothesis therefore is that the integrated clc element can excise from its chromosomal location and form a closed 'circular' intermediate. The closed intermediate can either reintegrate or be transferred to a new recipient cell, in which it can again integrate. Reintegration of the clc element has been observed in single cells which had an additional integration site engineered to induce GFP fluorescence expression when inserted by the clc element. ICEclc is behaving rather strangely: it becomes only active for transfer in a few percent of all cells in a population. This phenomenon is called bistability. Bistability of ICEclc can be observed by using transcriptional fusions of the integrase promoter to a gene for an autofluorescent protein.