Members of the genus Xenorhabdus are entomopathogenic bacteria that associate with nematodes. The nematode-bacteria pair infects and kills insects, with both partners contributing to insect pathogenesis and the bacteria providing nutrition to the nematode from available insect-derived nutrients. The nematode provides the bacteria with protection from predators, access to nutrients, and a mechanism of dispersal. Members of the bacterial genus Photorhabdus also associate with nematodes to kill insects, and both genera of bacteria provide similar services to their different nematode hosts through unique physiological and metabolic mechanisms. We posited that these differences would be reflected in their respective genomes. To test this, we sequenced to completion the genomes of Xenorhabdus nematophila ATCC 19061 and Xenorhabdus bovienii SS-2004. As expected, both Xenorhabdus genomes encode many anti-insecticidal compounds, commensurate with their entomopathogenic lifestyle. Despite the similarities in lifestyle between Xenorhabdus and Photorhabdus bacteria, a comparative analysis of the Xenorhabdus, Photorhabdus luminescens, and P. asymbiotica genomes suggests genomic divergence. These findings indicate that evolutionary changes shaped by symbiotic interactions can follow different routes to achieve similar end points.

The initial chemical steps in Tn5 transposition result in blunt end cleavage of the transposon from the donor DNA. We demonstrate that this cleavage occurs via a hairpin intermediate. The first step is a 3 hydrolytic nick by transposase. The free 3OH then attacks the phosphodiester bond on the opposite strand, forming a hairpin at the transposon end. In addition to forming precise hairpins, Tn5 transposase can form imprecise hairpins. This is the first example of imprecise hairpin formation on transposon end DNA. To undergo strand transfer, the hairpin must to be resolved by a transposase-catalyzed hydrolytic cleavage. We show that both precise and imprecise hairpins are opened by transposase. A transposition mechanism utilizing a hairpin intermediate allows a single transposase active site to cleave both 3 and 5 strands without massive protein/DNA rearrangements.Genomic DNA is known to undergo a variety of rearrangements, including inversions, deletions, duplications, and translocations. Transposition is one of the ways by which these rearrangements occur. The transposon Tn5 is a 5.8-kilobase pair prokaryotic, composite transposon consisting of two inverted repeats, IS50L and IS50R, that flank genes encoding antibiotic resistance. IS50R codes for the Tn5 transposase (Tnp), the protein catalyzing all the steps in transposition. Each IS50 repeat is bracketed by two different 19-bp 1 end sequences, termed the outside end (OE) and the inside end (IE), that are specifically recognized by Tn5 transposase ( Fig. 1) (reviewed in Refs. 1 and 2). Transposition of the full Tn5 element requires two OEs, whereas transposition of an IS50 element requires one OE and one IE. In vitro, Tn5 transposition requires only a hyperactive mutant transposase, such as EK54/LP372 Tnp, Mg 2ϩ , transposon DNA defined by two inverted 19-bp end sequences, and target DNA (3). Transposition frequency can be increased by using the mosaic end sequence (ME), a hyperactive, synthetic end sequence that is a hybrid of the OE and IE (Fig. 1) (4).Tn5 is a member of the "cut and paste" family of transposons that includes Tn10 and Tn7. Although the transposition reactions vary in details, they follow the same basic mechanism. Transposase binds to the transposon DNA at the end recognition sequences. Then, the end sequences are brought together via transposase oligomerization to form a complex nucleoprotein structure, 2 termed a synaptic complex (5, 6). Once a stable synaptic complex has been formed, the transposon end sequence-donor DNA boundary can be cleaved to release a pair of 3ЈOHs at the ends, which are then used in the next chemical step, strand transfer ( Fig. 2A). Strand transfer occurs via a one step transesterification (7,8) in which the 3ЈOHs attack phosphodiester bonds in the target DNA in a staggered fashion. For Tn5, the staggered attack leads to 9-bp gaps flanking the integrated transposon. The gaps are presumed to be filled in and ligated in the cell, and this leads to 9-bp duplications of the target sequence flanking the transposon (9, ...

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The gammaproteobacterium Xenorhabdus nematophila is a mutualistic symbiont that colonizes the intestine of the nematode Steinernema carpocapsae. nilB (nematode intestine localization) is essential for X. nematophila colonization of nematodes and is predicted to encode an integral outer membrane beta-barrel protein, but evidence supporting this prediction has not been reported. The function of NilB is not known, but when expressed with two other factors encoded by nilA and nilC, it confers upon noncognate Xenorhabdus spp. the ability to colonize S. carpocapsae nematodes. We present evidence that NilB is a surface-exposed outer membrane protein whose expression is repressed by NilR and growth in nutrient-rich medium. Bioinformatic analyses reveal that NilB is the only characterized member of a family of proteins distinguished by N-terminal region tetratricopeptide repeats (TPR) and a conserved C-terminal domain of unknown function (DUF560). Members of this family occur in diverse bacteria and are prevalent in the genomes of mucosal pathogens. Insertion and deletion mutational analyses support a beta-barrel structure model with an N-terminal globular domain, 14 transmembrane strands, and seven extracellular surface loops and reveal critical roles for the globular domain and surface loop 6 in nematode colonization. Epifluorescence microscopy of these mutants demonstrates that NilB is necessary at early stages of colonization. These findings are an important step in understanding the function of NilB and, by extension, its homologs in mucosal pathogens.

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