Excision of IS492 Requires Flanking Target Sequences and Results in Circle Formation in Pseudoalteromonas atlantica

Abstract

The gram-negative marine bacterium Pseudoalteromonas atlantica produces extracellular polysaccharide (EPS) that is important in biofilm formation by this bacterium. Insertion and precise excision of IS492 at a locus essential for extracellular polysaccharide production (eps) controls phase variation of EPS production in P. atlantica. Examination of IS492 transposition in P. atlantica by using a PCR-based assay revealed a circular form of IS492 that may be an intermediate in transposition or a terminal product of excision. The DNA sequence of the IS492 circle junction indicates that the ends of the element are juxtaposed with a 5-bp spacer sequence. This spacer sequence corresponds to the 5-bp duplication of the chromosomal target sequence found at all IS492insertion sites on the P. atlantica chromosome that we identified by using inverse PCR. IS492 circle formation correlated with precise excision of IS492 from the P. atlantica eps target sequence when introduced intoEscherichia coli on a plasmid. Deletion analyses of the flanking host sequences at the eps insertion site for IS492 demonstrated that the 5-bp duplicated target sequence is essential for precise excision of IS492 and circle formation in E. coli. Excision of IS492 inE. coli also depends on the level of expression of the putative transposase, MooV. A regulatory role for the circular form of IS492 is suggested by the creation of a new strong promoter for expression of mooV by the joining of the ends of the insertion sequence element at the circle junction.

Everyman's Guide to Bacterial Insertion Sequences

“…For these IS, the other end carries a −10 element oriented inwards towards the Tpase gene. Together with the −35, this generates a strong promoter on formation of the circle junction to drive the Tpase expression required for catalysis of integration (Chandler et al, this volume) (89)(90)(91). Hence, if integration occurs next to a resident −10 sequence, the IS −35 sequence can contribute to a hybrid promoter to drive expression of neighboring genes, see reference 92.…”

“…However, it remains to be determined whether they use a copy-paste mechanism or whether the circular intermediate is formed by excision. For example, IS492 was identified within the extracellular polysaccharide production (eps) gene of Pseudoalteromonas atlantica (90,235). IS492 clearly undergoes Tpase-dependent precise excision to regenerate a functional eps gene.…”

“…IS492 clearly undergoes Tpase-dependent precise excision to regenerate a functional eps gene. Like many other IS that use double-strand circular intermediate, circle formation results in the assembly of a junction promoter from a −35 promoter element in the right end oriented outwards and a −10 promoter element in the left end oriented inwards (90). In the case of IS1383, the −10 component of the promoter appears to lie within the DNA sequence located between the abutted IS ends.…”

“…In the case of IS1383, the −10 component of the promoter appears to lie within the DNA sequence located between the abutted IS ends. This promoter presumably serves to drive Tpase expression for the final integration step (90,234). For IS1383, amplification of a putative circle junction suggested that the abutted IS ends were separated by 10 bp composed of the 5 bp flanking each end in the original target site.…”

“…Excision of the IS regenerates the original target sequence (236). In the case of IS492, the flanking 5 bp were also essential for formation of the dsDNA circular form (90).…”