Chromosomal context directs high-frequency precise excision of IS492 in Pseudoalteromonas atlantica

Abstract

DNA rearrangements, including insertions, deletions, and inversions, control gene expression in numerous prokaryotic and eukaryotic systems, ranging from phase variation of surface antigens in pathogenic bacteria to generation of Ig diversity in human B cells. We report here that precise excision of the mobile element IS492 from one site on the Pseudoalteromonas atlantica chromosome directly correlates with phase variation of peripheral extracellular polysaccharide ((p)EPS) production from OFF (epsG::IS492) to ON (epsG(+)). In a previously undescribed application of quantitative PCR, we determined that the frequency of this transposase-dependent precise excision is remarkably high, ranging from 10(-3) to 10(-2) per cell per generation. High-frequency excision resulting in nonmutagenic repair of donor DNA is extremely unusual for classical transposable elements. Interestingly, high-frequency precise excision of IS492 does not occur at four different insertion sites on the P. atlantica chromosome, despite identity in the IS492 nucleotide sequences and 5- to 7-bp flanking DNA. The genome sequence revealed that epsG-associated IS492 is the only element inserted within a gene. Quantitative RT-PCR assays for externally derived transposase transcripts from each IS492 copy showed that IS492 at epsG has higher levels of host-initiated transcription through the element, suggesting that transcription per se or an increase in transposase (mooV) expression is responsible for the effect of chromosomal position on element excision. MooV levels and excision activity for IS492 inserted in forward and reverse orientations relative to plac and pT7 in Escherichia coli support that external transcription of mooV boosts transposase to a critical level required for detectable excision.

Fig. 1.

^pEPS phase variation in P. atlantica. Colony morphology switching between mucoid (^pEPS⁺) and crenated (^pEPS⁻) colonies is controlled by IS492 (white and cross-hatched bar) reversible insertion into a single site in epsG. Insertion and the precise excision of IS492 are mediated by the transposase, encoded by mooV (cross-hatched bar). The inserted element is flanked by a 5-bp direct repeat of the target sequence (black bar); precise excision of the element restores eps (9).

Fig. 2.

Correlation of IS492 precise excision from epsG, directly measured by qPCR, with ^pEPS phase variation in P. atlantica. (A) Two Taqman probes with different fluorophores (circles) and black hole quenchers (diamonds) are designed to separately measure amplification by the forward and reverse primers (arrows) of agrA (wavy line; base pairs 2849088–2850606) and restored epsG (dark gray line; base pairs 1303974–1304985). Release of the fluorophore (gray sun) from the probe by the 5′-3′ exonuclease activity of Taq polymerase fluorophore is measured (Bio-Rad iCycler Real Time PCR System). (B) The frequency of IS492 P_EX mean values are plotted vs. the frequency of P_PV mean values; the data are from six independent experiments.

Fig. 3.

Precise excision of IS492 from different P. atlantica chromosomal locations. Forward and reverse primers that flank IS492 at each insertion site were used in PCRs with chromosomal DNA template from HCD-1. For those reactions that yielded no PCR product (Direct PCR, Locus 1–4), the sensitivity of the PCR was increased by using aliquots of the Direct PCR assay with nested forward and reverse primers (Nested PCR). The predicted products for precise excision of copies 1–4 from their respective sites were generated and cloned into pCR2.1 (SI Table 2) for use as template to confirm the activity of the nested primers (Controls). The mobilities of the amplified unexcised IS492 elements with flanking DNA and of the amplified insertion sites for the precisely excised eps-associated element, and copies 3 and 4 are indicated. The DNA size marker (M) is the Promega (Madison, WI) 25-bp ladder.

Fig. 4.

qRT-PCR assay for host-initiated mooV transcripts. The curve-fit fluorescence units (CF RFU), adjusted for the signal base line, are plotted vs. the PCR cycle number from the qRT-PCRs with the cDNA generated from host-initiated mooV transcripts for each of the five IS492 copies on the P. atlantica chromosome (filled symbols) and for the reference trxA mRNA (open squares). The threshold bar determines the threshold cycle used to calculate the amount of starting template; the control reactions lacking reverse transcriptase (open circles) demonstrate the absence of detectable DNA contamination of the RNA preparations used. The results for RNA from crenated colonies at HCD (R-HCD1) are shown.

Fig. 5.

PCR assay for precise excision of IS492 under varying external transcription conditions in E. coli. Precise excision of IS492 is linked to the formation of a circularized IS492 (9). A PCR assay to detect circle junction (CJ) formation under different levels of external transcription through the element from upstream or downstream is depicted. IS492 (mooV) is in two orientations (A and B) between plac and pT7 on pCR2.1. PCR primers to detect CJ are shown as half arrows. Isopropyl β-d-thiogalactoside-inducible MooV is provided in trans from the expression vector, pAG900. The position of the PCR product is marked by an arrow. Western blot analysis revealed the relative expression of MooV under the different transcription conditions.