Fig 1.
Structural models of DUF490 and POTRA domains.
The colored regions under brackets were subjected to AlphaFold v2.0 modeling under default settings. All structures are oriented with the N-terminus down. A. AlphaFold v2.0 model of E. coli DUF490 (aa 923–1259 of TamB). B. AlphaFold v2.0 model of B. burgdorferi DUF490 (aa 1117–1443 of TamB) colorized to depict experimental segments dissected in this study: Segment 1 (aa 1117–1221) in blue, Segment 2 (aa 1222–1334) in magenta, and Segment 3 (aa 1335–1443) in wheat. C. AlphaFold v2.0 models of the DUF490 domains from B. burgdorferi (in blue) and E. coli (in green) superimposed. D. AlphaFold v2.0 model of B. burgdorferi BamA POTRA domains 1–5 colorized to depict individual POTRA domains: POTRA1 (aa 28–101) in cyan, POTRA2 (aa 102–180) in red, POTRA3 (aa 181–274) in orange, POTRA4 (aa 275–354) in green, and POTRA5 (aa 355–433) in yellow.
Fig 2.
In situ model of B. burgdorferi TamB DUF490 interaction with B. burgdorferi BamA.
The C-terminal BamA barrel is depicted in black and the N-terminal POTRA domains in green (POTRA1 labeled P1 and POTRA5 labeled P5). The DUF490 domain is depicted in three colors from the N-terminus: Segment 1 in blue (S1, aa 1117–1221), Segment 2 in magenta (S2, aa 1222–1334), and Segment 3 with the remainder of TamB (S3, aa 1335–1465) in wheat.
Fig 3.
B. burgdorferi DUF490 interacts with the POTRA domains of BamA.
Lower molecular weight bands (26 kDa) in anti-GST lanes correspond with the size of the GST tag. The black arrows next to blots indicate the GST fusion band and white arrows indicate the 6xHis fusion band. The colored arrows below the blots indicate proteins expressed in each assay and the expected molecular weights are listed here. A. Whole cell lysate and co-purification from E. coli expressing BbDUF490 (DUF, 66 kDa) as well as BbPOTRA1-5 (P1-5, 45 kDa), subjected to immunoblotting with anti-GST antibody for BbDUF490 and anti-6xHis antibody for BbPOTRA1-5. B. Whole cell lysate and co-purification from E. coli expressing BbDUF490 Segment 1 (S1, 39 kDa) as well as BbPOTRA1-5, subjected to immunoblotting with anti-GST antibody for Segment 1 and anti-6xHis antibody for BbPOTRA1-5. C. Whole cell lysate and co-purification from E. coli expressing BbDUF490 Segment 2 (S2, 39 kDa) as well as BbPOTRA1-5, subjected to immunoblotting with anti-GST antibody for Segment 2 and anti-6xHis antibody for BbPOTRA1-5. D. Whole cell lysate and co-purification from E. coli expressing BbDUF490 Segment 3 (S3, 39 kDa) as well as BbPOTRA1-5, subjected to immunoblotting with anti-GST antibody for Segment 3 and anti-6xHis antibody for BbPOTRA1-5. E. Whole cell lysate and co-purification from E. coli expressing OspC as well as BbPOTRA1-5, subjected to immunoblotting with anti-GST antibody for OspC (48 kDa) and anti-6xHis antibody for BbPOTRA1-5.
Fig 4.
B. burgdorferi DUF490 interacts with the POTRA2 and POTRA3 domains of BamA.
All samples were subjected to immunoblotting with anti-GST antibody for GST fusions and anti-6xHis antibody for BbPOTRA fusions. Lower molecular weight bands (26 kDa) in anti-GST lanes correspond with the size of the GST tag. The black arrows next to blots indicate the GST fusion band and white arrows indicate the 6xHis fusion band. The colored arrows below the blots indicate proteins co-expressed in each assay. A. Whole cell lysate and co-purification from E. coli expressing BbPOTRA1 (P1, 9 kDa) and BbDUF490 (66kD), BbDUF490 segments (39 kDa), or OspC (48 kDa). BbPOTRA1 is highlighted in the AlphaFold v2.0 model of BbPOTRA1-5 on the right. B. Whole cell lysate and co-purification from E. coli expressing BbPOTRA2 (P2, 9 kDa) and DUF490, DUF490 segments, or OspC. BbPOTRA2 is highlighted in the AlphaFold v2.0 model of BbPOTRA1-5 on the right. C. Whole cell lysate and co-purification from E. coli expressing BbPOTRA3 (P3, 11 kDa) and BbDUF490, BbDUF490 segments, or OspC. BbPOTRA3 is highlighted in the AlphaFold v2.0 model of BbPOTRA1-5 on the right. D. Whole cell lysate and co-purification from E. coli expressing BbPOTRA4 (P4, 9 kDa) and DUF490, BbDUF490 segments, or OspC. BbPOTRA4 is highlighted in the AlphaFold v2.0 model of BbPOTRA1-5 on the right. E. Whole cell lysate and co-purification from E. coli expressing BbPOTRA5 (P5, 9 kDa) and BbDUF490, BbDUF490 segments, or OspC. BbPOTRA5 is highlighted in the AlphaFold v2.0 model of BbPOTRA1-5 on the right.
Table 1.
DUF490 interacts with POTRA2 and POTRA3 in Borreliella.
Fig 5.
B. burgdorferi DUF490 interacts with BamB but not BamD.
Lower molecular weight bands (26 kDa) in anti-GST lanes correspond with the size of the GST tag. The black arrows next to blots indicate the GST fusion band and white arrows indicate the 6xHis fusion band. Bold arrows below the immunoblot images indicate proteins co-expressed in each experiment. A. Whole cell lysate and co-purification from E. coli expressing BbBamB (BamB, 37 kDa) and BbDUF490 (66 kDa), BbDUF490 segments (39 kDa), or OspC (48 kDa). All samples were subjected to immunoblotting with anti-GST antibody for GST fusions and anti-6xHis antibody for BbBamB. B. Whole cell lysate and co-purification from E. coli expressing BbBamD (BamD, 12 kDa) and BbDUF490 (66 kDa) or OspC (48 kDa). All samples were subjected to immunoblotting with anti-GST antibody for DUF490 or OspC and anti-6xHis antibody for BbBamD.
Fig 6.
Current working model of interaction between DUF490 and the BAM complex in Borreliella burgdorferi.
The B. burgdorferi TAM-BAM system based on combined interaction data and AlphaFold v2.0 modeling of each protein. BamA POTRA1-3 and DUF490 were modeled as a multimer and combined with the remainder of their structures. BamA is depicted in red, BamB depicted in blue, BamD depicted in yellow, TamB (aa 1–1116) depicted in mint green, and DUF490 (aa 1117–1443) depicted in dark green. The depicted membrane is a general representation of the borrelial membrane. Diacylated gray molecules represent phospholipids. Diacylated orange molecules represent glycolipids. Triacylated globular proteins represent other lipoproteins, pink squares represent metabolite transporters, green channels represent ABC transporters, and gray barrels represent porins. Created with BioRender.com.
Fig 7.
DUF490 and POTRA domains are structurally similar between species.
All structures are oriented with the N-terminus down. A. AlphaFold v2.0 model of S. Typhimurium DUF490 (StDUF490). B. AlphaFold v2.0 model of B. burgdorferi DUF490 (BbDUF490; red) aligned with S. Typhimurium DUF490 (StDUF490; blue) using PyMOL. C. AlphaFold v2.0 model of S. Typhimurium TamA POTRA1-3 (StTamAP1-3). D. AlphaFold v2.0 model of B. burgdorferi BamA POTRA1-5 (BbBamAP1-5; red) domains aligned with S. Typhimurium TamA POTRA1-3 domains (StTamAP1-3; blue) using PyMOL. E. AlphaFold v2.0 model of S. Typhimurium BamA POTRA1-5 (StBamAP1-5). F. AlphaFold v2.0 model of B. burgdorferi BamA POTRA2-4 (BbBamAP2-4; red) aligned with S. Typhimurium BamA POTRA2-4 (StBamAP2-4; blue) using PyMOL.
Fig 8.
Salmonella DUF490 interacts with the POTRA domains of both TamA and BamA.
Lower molecular weight bands (26 kDa) in anti-GST lanes correspond with the size of the GST tag. The black arrows next to blots indicate the GST fusion band and white arrows indicate the 6xHis fusion band. A. Whole cell lysate and co-purification from E. coli expressing one of the following: StDUF490 (DUF, 66 kDa) or Pal (42 kDa) as well as StTamA POTRA1-3 (P1-3, 31 kDa), subjected to immunoblotting with anti-GST antibody for GST fusions and anti-6xHis antibody for StTamAPOTRA1-3. B. Whole cell lysate and co-purification from E. coli expressing one of the following: StDUF490 (DUF, 66 kDa) or Pal (42 kDa) as well as StBamA POTRA1-5 (P1-5, 45 kDa), subjected to immunoblotting with anti-GST antibody for GST fusions and anti-6xHis antibody for StBamAPOTRA1-5. C. Whole cell lysate and co-purification from E. coli expressing StDUF490 (DUF, 66 kDa) and one of the following: StBamA POTRA1-5 (P1-5, 45 kDa) or OspC (22 kDa), subjected to immunoblotting with anti-GST antibody for StDUF490 and anti-6xHis antibody for StBamA POTRA1-5 and OspC.
Fig 9.
DUF490 interacts with BamA/TamA POTRA domains regardless of source species.
Lower molecular weight bands (26 kDa) in anti-GST lanes correspond with the size of the GST tag. The black arrows next to blots indicate the GST fusion band and white arrows indicate the 6xHis fusion band. A. Whole cell lysate and co-purification from E. coli expressing one of the following: BbDUF490 (DUF, 66 kDa) or OspC (48 kDa) as well as StTamA POTRA1-3 (P1-3, 31 kDa), subjected to immunoblotting with anti-GST antibody for GST fusions and anti-6xHis antibody for StTamA POTRA1-3. B. Whole cell lysate and co-purification from E. coli expressing one of the following: BbDUF490 (DUF, 66 kDa) or OspC (48 kDa) as well as StBamA POTRA1-5 (P1-5, 45 kDa), subjected to immunoblotting with anti-GST antibody for GST fusions and anti-6xHis antibody for StBamA POTRA1-5. C. Whole cell lysate and co-purification from E. coli expressing one of the following: StDUF490 (DUF, 66 kDa) or Pal (42 kDa) as well as BbBamA POTRA1-5 (P1-5, 45 kDa), subjected to immunoblotting with anti-GST antibody for GST fusions and anti-6xHis antibody for BbBamA POTRA1-5.
Fig 10.
Model of Salmonella OMP transport systems.
The Salmonella enterica serovar Typhimurium TAM-BAM system based on combined interaction data and AlphaFold v2.0 modeling of each protein. On the left, BamA is depicted in red, BamB in blue, BamC in magenta, BamD in yellow, BamE in orange, TamB in mint green, and DUF490 in dark green. StBamAPOTRA1-3 and StDUF490 were modeled as a multimer and combined with the remainder of their structures. On the right, TamA is depicted in red, TamB in mint green, and DUF490 in dark green. StTamAPOTRA1-3 were modeled in multimer mode with StDUF490. The depicted membrane is a general representation of a Salmonella membrane. Diacylated gray molecules represent phospholipids. Diacylated blue molecules represent lipopolysaccharide. Triacylated globular proteins represent other lipoproteins, pink squares represent metabolite transporters, tan channels represent ABC transporters, and gray barrels represent porins and other beta-barrel proteins. The PhoQ histidine kinase is depicted on the inner membrane and labeled. PhoP is represented as a pink rod shape, either phosphorylated (represented by the addition of a yellow circle with a P) or unphosphorylated. Created with BioRender.com.
Table 2.
Sequences and statistics for the Borreliella systemic model.
Table 3.
Sequences and statistics for the Salmonella systemic model.
Table 4.
Primers used in this study.