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Chromosomal Context Directs High-Frequency Precise Excision of IS492 in Pseudoalteromonas Atlantica

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Chromosomal Context Directs High-Frequency Precise Excision of IS492 in Pseudoalteromonas Atlantica Chromosomal context directs high-frequency precise excision of IS492 in Pseudoalteromonas atlantica Brian P. Higgins, Chandra D. Carpenter, and Anna C. Karls* Department of Microbiology, University of Georgia, 1000 Cedar Street, Athens, GA 30602-2605 Edited by Melvin I. Simon, California Institute of Technology, Pasadena, CA, and approved November 23, 2006 (received for review September 29, 2006) DNA rearrangements, including insertions, deletions, and inver- Insertion element IS492 controls peripheral extracellular poly- sions, control gene expression in numerous prokaryotic and eu- saccharide (pEPS) expression in the marine bacterium karyotic systems, ranging from phase variation of surface antigens Pseudoalteromonas atlantica by insertion and precise excision at a in pathogenic bacteria to generation of Ig diversity in human B single site within a predicted glucosyl-transferase gene (epsG) (Fig. cells. We report here that precise excision of the mobile element 1; see also refs. 8 and 9). IS492 is a member of the unusual IS110 IS492 from one site on the Pseudoalteromonas atlantica chromo- family of insertion elements that lack terminal inverted repeats and some directly correlates with phase variation of peripheral extra- encode novel recombinases that mediate transposition by an un- cellular polysaccharide (pEPS) production from OFF (epsG::IS492)to determined mechanism (10). The transposase of IS492, MooV, is ON (epsG؉). In a previously undescribed application of quantitative one of the defining members of the DEDD DNA recombinase PCR, we determined that the frequency of this transposase-depen- family, which includes the site-specific invertase Piv from Moraxella dent precise excision is remarkably high, ranging from 10؊3 to 10؊2 lacunata and Moraxella bovis, as well as the transposases of the IS110 per cell per generation. High-frequency excision resulting in non- family (11). MooV is required for precise excision of IS492 (9). mutagenic repair of donor DNA is extremely unusual for classical Although MooV and Piv appear to mediate conservative site- transposable elements. Interestingly, high-frequency precise exci- specific excision and inversion, respectively, these recombinases sion of IS492 does not occur at four different insertion sites on the show no identity or similarity to Y- or S-site-specific recombinases. P. atlantica chromosome, despite identity in the IS492 nucleotide Instead, the recombinases of the Piv-MooV family have a conserved sequences and 5- to 7-bp flanking DNA. The genome sequence DEDD motif, shown to be essential for Piv-mediated DNA inver- revealed that epsG-associated IS492 is the only element inserted sion, and are predicted to have a tertiary structural motif common within a gene. Quantitative RT-PCR assays for externally derived to the DDE transposases and the DEDD-motif Holliday junction transposase transcripts from each IS492 copy showed that IS492 at resolvases (RuvC-related; see refs. 12 and 13). epsG has higher levels of host-initiated transcription through the Many of the unusual features of IS492 and its transposition element, suggesting that transcription per se or an increase in products suggest a novel transposition mechanism, and here we transposase (mooV) expression is responsible for the effect of focus on the precise excision of IS492. We directly assayed chromosomal position on element excision. MooV levels and ex- precise excision from a single site within epsG and determine ϫ Ϫ2 cision activity for IS492 inserted in forward and reverse orienta- that the frequency of precise excision approaches 2 10 per tions relative to plac and pT7 in Escherichia coli support that cell per generation on solid medium, correlating well with the external transcription of mooV boosts transposase to a critical level frequency of colony-phase variation measured for the same cell populations. This level of precise excision from the chromosome required for detectable excision. is unprecedented for classical transposable elements (14, 15). We further show that the high frequency of IS492 precise excision is DEDD-motif recombinases ͉ IS110 family ͉ phase-variation frequency ͉ ͉ unique to the eps-associated copy of IS492 and positively cor- transposition quantitative PCR relates with the level of transcription that initiates upstream of the element and passes through the mooV gene. Both the ransposons are genetic elements that can move to multiple stimulation of excision by transcription through IS492 and the Tsites in a host genome and generate DNA rearrangements frequent repair of donor sequence in excision are unexpected that contribute to genome diversity. These mobile elements use features for a classical IS and strongly suggest that IS492 utilizes a variety of mechanisms for movement, including rolling circle a novel mechanism for transposition. transposition and nonreplicative transposition mediated by ty- rosine (Y)- and serine (S)-site-specific recombinases (1–4). Results However, the majority of the characterized transposons move by Precise Excision of IS492 from epsG in P. atlantica Occurs at an a common mechanism involving hydrolysis of phosphodiester Unusually High Frequency. It has been reported that P. atlantica bonds at element–host junctions and DNA strand transfer DB27 colony switching from crenated (pEPSϪ) to mucoid (pEPSϩ) (one-step transesterification) catalyzed by a classical DDE trans- posase (Asp, Asp, Glu catalytic motif). Although there are various intermediates in the transposition reactions of the clas- Author contributions: B.P.H., C.D.C., and A.C.K. designed research; B.P.H., C.D.C., and A.C.K. sical transposons, the chemistries of the DNA cleavage and performed research; A.C.K. contributed new reagents/analytic tools; B.P.H., C.D.C., and A.C.K. analyzed data; and B.P.H. and A.C.K. wrote the paper. strand-transfer reactions are conserved (reviewed in ref. 5). The authors declare no conflict of interest. Because the transposon is excised from donor DNA by hydrolysis GENETICS reactions, precise excision in which the host DNA is restored to This article is a PNAS direct submission. Ϫ ϩ the original target sequence is rarely observed. In eukaryotes, Abbreviations: pEPS, peripheral extracellular polysaccharide; pEPS , crenated pEPS ,mu- coid; PPV, frequency of phase variation; PEX, frequency of precise excision; HCD, high colony nonhomologous end-joining host functions can restore the donor density; LCD, low colony density; IS, insertion sequence; qPCR, quantitative PCR; SQ, sequence (6). In prokaryotes, composite transposons can be starting quantity; qRT-PCR, quantitative RT-PCR. excised precisely by a DNA replication-dependent process Data deposition: The sequence reported in this paper has been deposited in the GenBank (RecA- and transposase-independent) that utilizes the short database (accession no. NC 008228). direct repeats that flank the element and the long terminal *To whom correspondence should be addressed. E-mail: [email protected]. inverted repeats of the transposon (7). However, processes that This article contains supporting information online at www.pnas.org/cgi/content/full/ can result in precise excision of mobile elements are often 0608633104/DC1. mutagenic, generating deletions and small insertions. © 2007 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0608633104 PNAS ͉ February 6, 2007 ͉ vol. 104 ͉ no. 6 ͉ 1901–1906 Downloaded by guest on September 25, 2021 Methods). Precise excision of IS492 moves the primer to within 3 bp of the 5Ј end of the probe, which allows Taq polymerase to reach the probe and release FAM-490. The agr probe is positioned 2 bp from the forward primer, so that the HEX-530 fluorophore is released with every extension reaction. Representative data from one set of reactions, including the epsG and agrA standards, and the corresponding standard DNA plot for determining the quantities of agrA epsG Fig. 1. pEPS phase variation in P. atlantica. Colony morphology switching and restored in the DNA aliquots are shown in sup- between mucoid (pEPSϩ) and crenated (pEPSϪ) colonies is controlled by IS492 porting information (SI) Fig. 6. These template quantities are used (white and cross-hatched bar) reversible insertion into a single site in epsG. in calculating the PEX for each sample (see Materials and Methods). Insertion and the precise excision of IS492 are mediated by the transposase, Because the frequency of IS492 insertion is 3–4 orders of magni- encoded by mooV (cross-hatched bar). The inserted element is flanked by a tude lower than excision (B.P.H., unpublished data), the back rate 5-bp direct repeat of the target sequence (black bar); precise excision of the does not affect the calculation of precise excision frequency. element restores eps (9). The PPV and PEX values, determined for cells grown at HCD and LCD, fit a simple linear regression model with a r2 value of 0.945 and slope of 1.08 (Fig. 2B), indicating there is one precise excision is variable depending on growth conditions, including colony den- event for each colony-morphology phase-variation event. Multiple sity on marine agar (MA; ref. 8). Therefore, in our determination regression analyses show that PEX and PPV are strongly correlated of the frequencies of precise excision of IS492 from epsG (PEX) and with each other, and this relationship is independent of colony pEPSϪ to pEPSϩ phase variation (P )inP. atlantica, we used equal PV density (SI Text). The average PEX and PPV at LCD are 1.2 Ϯ 0.5 ϫ numbers of cells from colonies plated at low-colony density (LCD) 10Ϫ2 and 1.0 Ϯ 0.5 ϫ 10Ϫ2 per cell per generation, respectively, and and high-colony density (HCD) on MA. at HCD, they are 1.2 Ϯ 0.8 ϫ 10Ϫ3 and 1.3 Ϯ 0.6 ϫ 10Ϫ3 per cell We developed a multiplex quantitative PCR (qPCR) assay to per generation, respectively. directly measure both the total number of chromosomes and the number in which IS492 has precisely excised from the epsG site (Fig. IS492 Copies at Locations Other Than the eps Site on the P.
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