TABLE 3.
Summary of yeast and translocation assay resultsa
| A33 locus tag or ORFb | Productc | Yeast inhibition without stressorsd,e | % of HeLa cells showing translocatione,f | Effector proteine,g |
|---|---|---|---|---|
| A33_1662 | HP (VopE) | ++ | 3 | Y |
| A33_1663 | HP (VopX) | +++ | 30 | Y |
| A33_1665 | CHP | + | Y/N | N |
| A33_1667 | CHP | N | NT | — |
| A33_1668 | HP | + | 0 | N |
| A33_1671 | CHP | N | NT | — |
| A33_1674 | HP | N | 0.06 | Nh |
| A33_1677 | HP | N | NT | — |
| A33_1678 | HP (VopH) | N | 0.02 | Y |
| A33_1679 | HP | N | NT | — |
| A33_1680 | CHP (VopA) | ++ | 0.05 | Y |
| A33_1683 | HP | N | NT | — |
| A33_1684 | HP (VopM) | + | 31 | Y |
| A33_1685 | CHP | N | NT | — |
| A33_1687 | CHP (VopI) | + | 0.03 | Y |
| A33_1688 | CHP | N | NT | — |
| A33_1690 | HP (VopW) | N | 0.08 | Y |
| A33_1691 | HP | N | NT | — |
| A33_1694 | CHP | N | NT | — |
| A33_1695 | HP | + | 0 | N |
| A33_1696 | VopF | ++++ | ∼0.05 (PC) | Y |
| A33_1697 | HP (VopG) | + | 11 | Y |
| A33_1699 | HP (VopK) | ++++ | 0.23 | Y |
| A33_1700 | CHP (VopY) | N | 0.13 | Y |
| A33_1703 | CHP | N | 0 | N |
| A33_1704 | HP (VopZ) | N | 7 | Y |
| ORF58 | HP | N | NT | — |
| A33_1706 | CHP | ++++ | 0 | N |
Twenty-seven candidate ORFs were tested in yeast for their ability to inhibit growth. Those showing growth inhibition were then screened for translocation using a FRET-based assay as described in the text. ORFs are listed as their A33 locus tag, except for ORF58, which was identified by our own annotation.
Our annotation of the AM-19226 T3SS island identified alternative start sites for some genes. Differences compared to the NCBI annotation are noted as follows. A33_1665 has an additional 243 bp upstream of the NCBI annotation. A33_1668 has an additional 160 bp upstream of the NCBI annotation. A33_1671 has an additional 15 bp upstream of the NCBI annotation. A33_1677 has an additional 6 bp upstream of the NCBI annotation. A33_1679 has an additional 45 bp upstream of the NCBI annotation. A33_1680 has an additional 39 bp upstream of the NCBI annotation. Due to DNA sequence differences, our annotation predicts that A33_1683 starts 12 bp upstream of the NCBI annotation; our annotation is consistent with the slightly larger protein annotated for Vibrio parahaemolyticus strain RIMD2210633. A33_1685 has an additional 465 bp upstream of the NCBI annotation. A33_1687 has an additional 21 bp upstream of the NCBI annotation. A33_1691 begins 66 bp downstream of the NCBI-annotated translational start site. A33_1694 has an additional 36 bp upstream of the NCBI annotation. A33_1695 has an additional 9 bp upstream of the NCBI annotation. A33_1703 has an additional 60 bp upstream of the NCBI annotation. A33_1704 has an additional 33 bp upstream of the NCBI annotation. ORF58 is our annotation of a 255-bp ORF; A33_1705 is annotated by NCBI as a 120-bp ORF in the same region but does not overlap with ORF58.
ORFs that we designated as effector proteins were assigned “Vop” names as shown in parentheses. HP, hypothetical protein; CHP, conserved hypothetical protein.
Growth defects were scored as mild (+), moderate (++), strong (+++), and severe (++++).
Y, yes; N, no.
Y/N, translocation was variable; NT, not tested; PC, positive control.
Effector protein designation (yes/no) was based on the results of the translocation assay. —, ORF was not tested for translocation.
A33_1674 is translocated independently of the T3SS.