Virulence properties of cariogenic Streptococcus mutans depend on integral membrane proteins. Bacterial cotranslational protein trafficking involves the signal recognition particle (SRP) pathway components Ffh and FtsY, the SecYEG translocon, and YidC chaperone/insertases. Unlike Escherichia coli, S. mutans survives loss of the SRP pathway and has two yidC paralogs. This study characterized YidC1 and YidC2 interactomes to clarify respective functions alone and in concert with the SRP and/or Sec translocon. Western blots of formaldehyde cross-linked or untreated S. mutans lysates were reacted with anti-Ffh, anti-FtsY, anti-YidC1, or anti-YidC2 antibodies followed by mass spectrometry (MS) analysis of gel-shifted bands. Cross-linked lysates of wild-type and ΔyidC2 strains were reacted with anti-YidC2-coupled Dynabeads, and cocaptured proteins were identified by MS. Last, YidC1 and YidC2 C-terminal tail-captured proteins were subjected to two-dimensional (2D) difference gel electrophoresis and MS analysis. Direct interactions of putative YidC1 and YidC2 binding partners were confirmed by bacterial two-hybrid assay. Our results suggest YidC2 works preferentially with the SRP pathway, while YidC1 is preferred for SRP-independent Sec translocon-mediated translocation. YidC1 and YidC2 autonomous pathways were also apparent. Two-hybrid assay identified interactions between holotranslocon components SecYEG/YajC and YidC1. Both YidC1 and YidC2 interacted with Ffh, FtsY, and chaperones DnaK and RopA. Putative membrane-localized substrates HlyX, LemA, and SMU_591c interacted with both YidC1 and YidC2. Identification of several Rgp proteins in the YidC1 interactome suggested its involvement in bacitracin resistance, which was decreased in ΔyidC1 and SRP-deficient mutants. Collectively, YidC1 and YidC2 interactome analyses has further distinguished these paralogs in the Gram-positive bacterium S. mutans. IMPORTANCE Streptococcus mutans is a prevalent oral pathogen and major causative agent of tooth decay. Many proteins that enable this bacterium to thrive in its environmental niche and cause disease are embedded in its cytoplasmic membrane. The machinery that transports proteins into bacterial membranes differs between Gram-negative and Gram-positive organisms, an important difference being the presence of multiple YidC paralogs in Gram-positive bacteria. Characterization of a protein’s interactome can help define its physiological role. Herein, we characterized the interactomes of S. mutans YidC1 and YidC2. Results demonstrated substantial overlap between their interactomes but also revealed several differences in their direct protein binding partners. Membrane transport machinery components were identified in the context of a large network of proteins involved in replication, transcription, translation, and cell division/cell shape. This information contributes to our understanding of protein transport in Gram-positive bacteria in general and informs our understanding of S. mutans pathogenesis.

20Virulence properties of cariogenic Streptococcus mutans depend on integral membrane proteins. 21Bacterial protein trafficking involves the co-translational signal recognition particle (SRP) 22 pathway components Ffh and FtsY, the SecY translocon, and membrane-localized YidC 23 chaperone/insertases. Unlike Escherichia coli, S. mutans survives loss of the SRP pathway. In 24 addition, S. mutans has two yidC paralogs. The yidC2 phenotype largely parallels that of ffh 25 and ftsY while the yidC1 phenotype is less severe. This study defined YidC1 and YidC2 26 interactomes to identify their respective functions alone and in concert with the SRP, ribosome, 27 and/or Sec translocon. A chemical cross-linking approach was employed, whereby whole cell 28 lysates were treated with formaldehyde followed by Western blotting using anti-Ffh, FtsY, 29 YidC1 or YidC2 antibodies and mass spectrometry (MS) analysis of gel-shifted bands. Cross-30 linked lysates from WT and yidC2 strains were also reacted with anti-YidC2 antibodies 31 coupled to magnetic Dynabeads TM , with co-captured proteins identified by MS. Additionally, C-32 terminal tails of YidC1 and YidC2 were engineered as glutathione-S-transferase fusion proteins 33 and subjected to 2D Difference Gel Electrophoresis and MS analysis after being reacted with 34 non-cross-linked lysates. Results indicate that YidC2 works in concert with the SRP-pathway, 35 while YidC1 works in concert with the SecY translocon independently of the SRP. In addition, 36 YidC1 and/or YidC2 can act alone in the insertion of a limited number of small integral 37 membrane proteins. The YidC2-SRP and YidC1/SecY pathways appear to function as part of an 38 integrated machinery that couples translation and transport with cell division, as well as 39 transcription and DNA replication. 40 41 42 Importance 43