PMC

(A) Interaction data are from motility arrays (E. coli) or genome-wide screens (in Treponema pallidum) as validated by homologous interactions (“interologs”) in other species (black). Interactions in open segments do not have interologs in other species. All interologs have been shown experimentally and have been derived from MPIDB []. (B) Interactions data from motility array screens were classified into one of three classes: “known”, plausible, and unclear (unknown). Most interactions (34% + 23% = 57%) detected with pDEST22/pDEST32 were either known or plausible while only 34% (14+20%) of the interactions detected with pGBKT7/pGADT7 were assigned to this class (see ).

All fusions are based on Gal4 activation domain (AD, preys: A) and Gal4 DNA binding domains (DBD, baits: B). Prey fusions mainly differ by the size of the linker between the AD and the prey ORF and the C-terminal peptide fused to these ORFs. NLS = nuclear localization signal. The amino acid sequence of the linker region between Gal4 AD or DBD and the prey or bait ORF is indicated above each fusion. Similarly, the C-terminal peptide derived from the vector is shown. All loxP-containing vectors (pLP-GADT7, pLPGBKT7, pAS1-LP) were used with ORFs that carried their endogenous stop codons, so they do not contain any C-terminal peptide.

(A) Number of interactions with individual baits found in T. pallidum whole-genome screens with pAS1-LP and pLP-GBKT7 bait constructs. On average, pAS1 baits produce more interactions than pLP-GBKT7 baits. (B) Number of interactions in E. coli motility array screens, plotted for individual baits. Note that all pDEST32 baits were tested against 90 pDEST22 preys; similarly, all pGBKT7g baits were tested against pGADT7g preys. pGBKT7g baits generally show more interactions than pDEST32. (C,D) Overlapping interactions between different datasets. (C) Overlap between the total numbers of interactions from 49 screens using motility proteins as baits (in bait vectors pLP-GBKT7 and pAS1-LP) against the whole-genome T. pallidum array. (D) Overlap between E. coli motility array screens using bait/prey vector pairs pGBKT7g/pGADT7g and pDEST32/pDEST22. Note that exactly the same set of proteins pairs (i.e. E.coli flagellum proteins) was tested. 24 published interactions among E. coli flagellar proteins (from MPIDB []) are included as gold-standard dataset. Note that despite the significant difference in total interactions, the overlap with the gold-standard set is very similar. (E,F) Fraction of baits that yielded interaction data in each of the whole genome (E) or motility array screens (F). For example, in the whole-genome screens in T. pallidum, 20% of all baits yielded interactions only as pAS1-LP baits, while 35% of all baits yielded interactions with both pAS1-LP and pLP-GBKT7. Note that the overlap between pAS1-LP and pLP-GBKT7 (E) was significantly larger than between the pDEST32/pDEST22 and pGBKT7g/pGADT7g pairs (F).

(A) Treponema pallidum whole-genome array screen as described in []. All T. pallidum ORFs were expressed in pLP-GADT7g and screened with 49 T. pallidum flagellum baits cloned into pLP-GBKT7 and pAS1-LP bait vectors. Two out of 98 screens (2 x 49) are shown in the top bar; a single plate (out of 11) is shown enlarged. Left: pLP-GBKT7-FliC (ORF TP0870), right: pAS1-LP-FliC (ORF TP0870). (B) E. coli motility-specific prey arrays expressed only 96 known and predicted motility-related prey proteins expressed in the pGADT7g (left) and pDEST32 (right) prey vectors. In contrast to (A), both bait and prey vectors were different although the protein pairs in the left and right panels are exactly the same. That is, except that the left panel has protein pairs expressed in pGBKT7g/pGADT7g vectors and the right panel in pDEST32/pDEST22 vectors. Shown here are screens with E. coli FliA, an RNA polymerase sigma factor for flagellar operons, that was screened against all 90 E. coli motility preys. The pDEST32/pDEST22 pairs show markedly different and overlapping interactions compared to the pGBKT7g/pGADT7g pairs. Strong and reproducible interactions are labeled, weak and potentially spurious interactions in the pGBKT7g/pGADT7g panel are not labeled