TABLE 1.
Genes of JCVI-syn2.0 that show a skew in the orientation of transposon insertionsa
| Beg | End | Direction | Preferred insertion orientation | RGD fragment | Length (aa) | Locus tag | Gene HSW 150126 | P1F | P1R | P1F:R | P2F | P2R | P2F:R | P6F | P6R | P6F:R | Likely reason | Flanking gene affected | New context in syn2.0? | Current annotation | Functional classification | System |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 2675 | 3217 | f | f | 1 | 180 | _0003 | i | 5 | 0 | 1 | 6 | 3 | 0.33 | 1 | 0 | 0.99 | Upstream train wreck avoided | (_0002) DNA Pol III beta subunit | No | RNase MS | Equivalog | Ribosome biogenesis |
| 55909 | 56499 | r | r | 1 | 196 | _0046 | n | 0 | 12 | 1 | 3 | 13 | 0.62 | 0 | 4 | 1 | Downstream read-through needed | (_0045) thymidylate kinase | No | Recombination protein | Equivalog | DNA repair |
| 63727 | 64701 | f | f | 1 | 324 | _0063 | n | 22 | 7 | 0.52 | 53 | 12 | 0.63 | 22 | 2 | 0.83 | Downstream read-through needed | (_0064) lysyl tRNA synthetase | No | Uncharacterized tRNA dihydrouridine synthase | Generic | tRNA modification |
| 74697 | 75536 | r | r | 1 | 279 | _0077 | n | 1 | 9 | 0.8 | 3 | 27 | 0.8 | 0 | 4 | 1 | Downstream read-through needed | (_0076) asparaginal tRNA synthetase | No | Low-specificity hydrolase | Putative | Unclear |
| 77752 | 77973 | r | r | 1 | 73 | _0080 | n | 0 | 6 | 1 | 2 | 10 | 0.67 | 0 | 2 | 1 | ? | ? | No | Uncharacterized protein | Unknown | Unclear |
| 87708 | 88820 | r | r | 2 | 370 | _0108 | i | 3 | 15 | 0.67 | 2 | 12 | 0.71 | 0 | 2 | 1 | Downstream read-through needed | (_0107) nusB transcription antitermination | No | Lipoprotein, putative | Generic | Lipoprotein |
| 91384 | 192304 | r | f | 2 | 306 | _0114 | i | 4 | 1 | 0.6 | 6 | 2 | 0.5 | 0 | 0 | 0 | Downstream train wreck avoided | (_0113) glycosyl transferase, group 2 | No | Glycosyltransferase, group 2 family protein | Putative | Lipid salvage and biogenesis |
| 101419 | 102480 | f | r | 2 | 353 | _0132 | i | 0 | 3 | 1 | 0 | 7 | 1 | 0 | 0 | 0 | Downstream read-through lethal | (_0133) toxin-peptidase, S8/S53 family | No | Toxin-antitoxin AAA ATPase | Probable | Toxin-antitoxin |
| 125069 | 125563 | r | r | 2 | 164 | _0164 | i | 0 | 6 | 1 | 1 | 4 | 0.6 | 0 | 0 | 0 | Downstream read-through needed | (_0163) alanyl tRNA synthetase | No | Hypothetical protein | Unknown | Unclear |
| 204874 | 205368 | r | r | 3 | 164 | _0301 | i | 2 | 6 | 0.5 | 0 | 1 | 0.99 | 0 | 0 | 0 | Downstream read-through needed | (_0300) nusA transcription termination/antitermination | No | rimP | Putative | Ribosome biogenesis |
| 294566 | 297532 | r | r | 4 | 988 | _0415 | i | 1 | 6 | 0.71 | 1 | 3 | 0.5 | 0 | 0 | 0 | ? | ? | No | Chromosome segregation protein SMC | Equivalog | Chromosome segregation |
| 468223 | 469179 | r | r | 7 | 318 | _0697 | n | 4 | 11 | 0.47 | 3 | 5 | 0.25 | 0 | 8 | 1 | Downstream read-through needed | (_0696) RDD family protein | Yes (upstream) | Glycosyltransferase, group 2 family protein | Generic | Unclear |
| 477490 | 478326 | f | f | 7 | 278 | _0728 | i | 15 | 5 | 0.5 | 6 | 1 | 0.71 | 0 | 0 | 0 | Downstream read-through needed | (_0729) phosphoglycerate mutase | No | HAD hydrolase, family IIB | Generic | Unclear |
| 483396 | 484049 | f | f | 7 | 217 | _0747 | i | 9 | 1 | 0.8 | 0 | 2 | 1 | 0 | 0 | 0 | ? | ? | Yes (up- and downstream) | punA | Probable | Nucleotide salvage |
| 506742 | 507185 | r | r | 8 | 147 | _0800 | i | 1 | 9 | 0.8 | 1 | 0 | 0.99 | 0 | 0 | 0 | Downstream read-through needed | (_0799) glyA, serine hydroxymethyltransferase | Yes (upstream) | rpiB: ribose 5-phosphate isomerase B | Equivalog | Metabolic process |
| 514997 | 516172 | r | r | 8 | 391 | _0805 | i | 2 | 18 | 0.8 | 0 | 6 | 1 | 0 | 2 | 1 | Downstream read-through needed | (_0804) RNA Pol, beta subunit | No | Transcription factor | Generic | Regulation |
Data correspond to the indicated 16 genes that show a skew in the orientation of viable Tn5-Puror insertions. The coordinates of each gene on the JCVI-syn2.0 genome (GenBank accession no. CP014992.1) are listed in the “Beg” (beginning) and “End” columns. Columns P1F, P2F, and P6F list the numbers of different insertion sites for Tn5-Puror in a particular gene in the “forward orientation” observed for passages 1, 2, and 6. The analogous numbers of insertion sites in the “reverse orientation” are shown in columns P1R, P2R, and P6R. Skew values were calculated for each gene at generations 1, 2, and 6 as described in the text, and data are listed as PnF:R (where “n” represents the passage number). For example, the skew value for gene _0046 in passage 2 is P2F:R = ABS (3 − 14)/16 = 0.62. A likely reason for the observed skew is listed where the data suggest one (see column “Likely reason”). The flanking gene whose functionality likely depends on the orientation of the Tn5-Puror insertion is indicated in the “Flanking gene affected” column. If the context of the gene in JCVI-syn2.0 is different from that in JCVI-syn1.0, due to removal of flanking genes, then this is indicated. Information concerning the annotation for each listed gene is shown in the last three columns, “Current annotation,” “Functional classification,” and “System” (updated from a previously described annotation [2]). aa, amino acids; Equivalog, gene (or corresponding encoded protein) that is known to have been conserved in function since the last common ancestral sequence; Pol III, polymerase III; RGD, Arg-Gly-Asp.