GIII 34 35 36 41 42 43 51 52 53 69 70 71 72 64 65 66 67 68 77 78 79 104 105 GII 18 19 20 21 22 © 2010712 1Lande9s8.21 ± 0.0B7 72 TT-AA 108 1 109 19 110 54 111 17 112 4 are distantly related to the IS200 family of Number of occurrences REPIN REP conservation within insertion sequences and which are typically within SBW25 orientationa REPIN (%) ± std devb flanked by multiple REPINs. 1 AA-TT 94.56 ± 0.4 There are several notable features of 11 RAYTs and their associated REPINs. First, 3 RAYT-encoding genes are typically short 6 98.22 ± 0.06 (~500 bp), and while highly conserved 13 AA-TT residues are apparent5 [among which are 9 the HUH and Y motifs, which are 1 94.34 ± 0.7 essential for transposition by TnpA of 8 AA-TT IS60512 (a member of the IS200 family)], 12 overall, the genes show substantive divers- 9 TT-AA 97.19 ± 0.8 ity (e.g., 22 RAYTs from the sequenced 11 138 Pseudomonas genomes show just 57% and 59 53% pairwise identity at the nucleotide 2 TT-AA 89.52 ± 1.2 level and amino acid level, respectively). 1 Second, each RAYT has a specific asso- 4 ciation with a particular sequence type of 0 REPIN. This is evident in the genome of 5 SBW25, which harbours three distinct i ogenosmeis cassociiated ewith nspeccific a faemily . 4 94.32 ± 1.3 RAYTs, each RAYT in the SBW25 (GI, GII or GIII) of REPINs. Third, TT-AA RAYTs are only ever present as single copy entities (in those genomes harbouring more than a single RAYT each RAYT is Do 7not distribin uSBWt25 aereas d.ifferent from each other 3 97.63 ± 0.06 distinctly different, e.g., the three RAYTs 7 TT-AA as they are from any RAYT chosen at 4 random from the total population of 1 Pseudomonas RAYTs). This last fact is 2 TT-AA 99.43 ± 1e-12 particularly curious, because it begs an 128 explanation for the maintenance of RAYTs in bacterial genomes. 180 TT-AA 95.21 ± 1.0 The raison d’etre for transposons and related elements is to disproportionately increase their representation within a given host genome (and to disseminate hori- zontally wherever possible).14,15 However, extinction is the long-term fate of most transposons given that selection is rela- tively impotent when it comes to purging REPIN diversity becomes more com- before in E. coli,13 but no mechanistic or deleterious mutations in transposases. This plex when the focus shifts to higher order evolutionary explanation for their forma- is because transposons encoding defective arrangements (multiple REPINs in close tion has been put forward. transposases (non-autonomous transpo- proximity). In SBW25 there are various sons) can exploit transposase function structurally different arrangements. The REPINs and RAYTs encoded by functional (autonomous) least common arrangement consists of transposons. The weakness of purifying co-localized REPINs. Such co-localized REPINs, if mobile elements, as suggested, selection means that non-autonomous REPINs are no more frequently found are too short to encode their own trans- elements are expected to increase in than expected by random chance. The position machinery. Their movement frequency—even to the point where they most common arrangement consists of thus requires exploitation of transposase may drive the functional family extinct.16 highly structured tandemly repeated activity encoded by some other fully The fact that RAYTs are present as just REPINs.10 In SBW25 these comprise as autonomous element. Strongly implicated single copy entities is indicative of their many as five tandem REPIN repeats. Such are the so-named REP-associated tyrosine incapacity for transposition. If RAYTs organisations have also been observed transposases (RAYTs): transposases that cannot transpose, then selection cannot aShows the two bases that are observed in the center of each palindrome (either AA or TT see Table 1) contained within a REPIN. bAverage pairwise identity and standard deviation of the 16 bp long REP sequences (Table 1) that are found as part of REPINs. Analyses are based on methods applied to REP conservation studies performed in our earlier paper.10 Table 2. Characteristics of REPINs found in the SBW25 genome REPIN GI Distance between REPs 264 Mobile Genetic Elements Volume 1 Issue 4