Membrane proteomic analysis reveals overlapping and independent functions of Streptococcus mutans Ffh, YidC1, and YidC2

Abstract

A comparative proteomic analysis was utilized to evaluate similarities and differences in membrane samples derived from the cariogenic bacterium Streptococcus mutans, including the wild‐type strain and four mutants devoid of protein translocation machinery components, specifically ∆ffh, ∆yidC1, ∆yidC2, or ∆ffh/yidC1. The purpose of this work was to determine the extent to which the encoded proteins operate individually or in concert with one another and to identify the potential substrates of the respective pathways. Ffh is the principal protein component of the signal recognition particle (SRP), while yidC1 and yidC2 are dual paralogs encoding members of the YidC/Oxa/Alb family of membrane‐localized chaperone insertases. Our results suggest that the co‐translational SRP pathway works in concert with either YidC1 or YidC2 specifically, or with no preference for paralog, in the insertion of most membrane‐localized substrates. A few instances were identified in which the SRP pathway alone, or one of the YidCs alone, appeared to be most relevant. These data shed light on underlying reasons for differing phenotypic consequences of ffh, yidC1 or yidC2 deletion. Our data further suggest that many membrane proteins present in a ∆yidC2 background may be non‐functional, that ∆yidC1 is better able to adapt physiologically to the loss of this paralog, that shared phenotypic properties of ∆ffh and ∆yidC2 mutants can stem from impacts on different proteins, and that independent binding to ribosomal proteins is not a primary functional activity of YidC2. Lastly, genomic mutations accumulate in a ∆yidC2 background coincident with phenotypic reversion, including an apparent W138R suppressor mutation within yidC1.

Protein Interactomes Identify Distinct Pathways forStreptococcus mutansYidC1 and YidC2 Membrane Protein Insertases

“…The SRP pathway is dispensable in S. mutans , although its disruption results in growth impairment, environmental stress-sensitivity, and diminished genetic competence [6] [7]. Deletion of S. mutans yidC2 causes a similar phenotype, whereas deletion of yidC1 appears less detrimental [7-10]. YidC is essential in E. coli [11].…”

“…Nor is a double yidC2 / ffh deletion mutant. In contrast, an ffh/yidC1 mutant is viable, albeit severely stress sensitive and growth impaired [10]. These results suggest synthetic lethality and functional redundancies between the SRP pathway and YidC2, and between YidC1 and YidC2.…”

“…While YidC1 and YidC2 apparently substitute for one another in some cases, distinct functional activities have been identified. YidC1 impacts cell surface biogenesis and bacterial adhesion more than YidC2, while YidC2 impacts cell wall biosynthesis and localization of penicillin binding proteins to the division septum [9, 10]. YidC1 and YidC2 demonstrate 27% amino acid homology and 48% similarity, each having six predicted transmembrane (TM) domains in the preprotein, and five in the mature insertase (TM2-TM6).…”

“…The cytoplasmic C-terminal tail of YidC2 is longer and more highly charged than YidC1’s, and appending the YidC2 tail onto YidC1 enables the chimeric protein to partially complement ΔyidC2 stress sensitivity [8]. Furthermore, gain of YidC2-like function point mutations have been reported within TM2 of S. mutans YidC1 [10], as well as within TM2 of SpoIIIJ, the YidC1 homolog of Bacillus subtilis [12]. Thus, while there is functional overlap between Gram-positive dual YidCs, paralog-specific features are recognized.…”

“…Respective substrates of integrated YidC/SecYEG and YidC/SecYEG/SRP pathways are largely unknown. Comparison of the membrane proteomes of S. mutans wildtype and mutant strains lacking ffh, yidC1, yidC2, or ffh/yidC1 suggested that its SRP pathway works in concert with YidC1 or YidC2 specifically, or with no preference, to insert most membrane-localized substrates [10]. In a few instances only the SRP pathway, or only YidC1 or YidC2, appeared to be required [10].…”