YlxM Is a Newly Identified Accessory Protein That Influences the Function of Signal Recognition Particle Pathway Components in Streptococcus mutans

Abstract

Streptococcus mutans is a cariogenic oral pathogen whose virulence is determined largely by its membrane composition. The signal recognition particle (SRP) protein-targeting pathway plays a pivotal role in membrane biogenesis. S. mutans SRP pathway mutants demonstrate growth defects, cannot contend with environmental stress, and exhibit multiple changes in membrane composition. This study sought to define a role for ylxM, which in S. mutans and numerous other bacteria resides directly upstream of the ffh gene, encoding a major functional element of the bacterial SRP. YlxM was observed as a produced protein in S. mutans. Its predicted helix-turn-helix motif suggested that it has a role as a transcriptional regulator of components within the SRP pathway; however, no evidence of transcriptional regulation was found. Instead, capture enzyme-linked immunosorbent assay (ELISA), affinity chromatography, and bio-layer interferometry (BLI) demonstrated that S. mutans YlxM interacts with the SRP components Ffh and small cytoplasmic RNA (scRNA) but not with the SRP receptor FtsY. In the absence of FtsY, YlxM increased the GTP hydrolysis activity of Ffh alone and in complex with scRNA. However, in the presence of FtsY, YlxM caused an overall diminution of net GTPase activity. Thus, YlxM appears to modulate GTP hydrolysis, a process necessary for proper recycling of SRP pathway components. The presence of YlxM conferred a significant competitive growth advantage under nonstress and acid stress conditions when wild-type and ylxM mutant strains were cultured together. Our results identify YlxM as a component of the S. mutans SRP and suggest a regulatory function affecting GTPase activity.

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

“…YidC2 works in concert with the signal recognition particle (SRP) pathway. The SRP is comprised of Ffh, a small cytoplasmic RNA, and the YlxM accessory protein present only in Gram-positive bacteria [63]. The SRP targets the ribosome nascent chain complex to the membrane via a reversible interaction of Ffh with the SRP receptor FtsY.…”

“…Cell lysate samples were electrophoresed on 4-20% precast gels (Bio-Rad Laboratories, Hercules, CA) in Tris-Glycine-SDS buffer. Replicate gels were stained with Coomassie Blue R 250 or transblotted onto Immobilon polyvinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories, Hercules, CA), reacted with affinity-purified YidC1 or YidC2 C-terminal-specific polyclonal rabbit antibodies (1:1000) [26], or anti-Ffh or anti-FtsY polyclonal rabbit antisera (1:1000) [63], followed by horseradish peroxidase-labeled anti-rabbit IgG (MP Biomedicals, Irvine, CA) (1:5000), and developed using the enhanced-chemiluminescence (ECL) Western blotting system (GE Healthcare).…”

Breaking the bacterial protein targeting and translocation model: oral organisms as a case in point

“…Further, repressing HBsu increased scRNA production and led to precursor protein accumulation, showing an important function of HBsu in the SRP targeting pathway (Yamazaki et al ., ). Similarly, an SRP accessory protein was recently identified in S. mutans (Williams et al ., ). The gene encoding YlxM is located directly upstream of ffh in S. mutans , a conserved gene organization in most gram‐positive and some gram‐negative bacteria.…”

“…A helix‐turn‐helix protein, YlxM, was predicted to be a DNA‐binding protein and suspected to regulate SRP component transcription/translation (Samuelsson et al ., ; Oganesyan et al ., ). Surprisingly, functional studies showed no evidence of gene regulatory function, but rather a direct interaction between YlxM and both Ffh and the scRNA (Williams et al ., ). In addition, YlxM increased GTP hydrolysis of Ffh.…”

A Systematic Proteomic Analysis of Listeria monocytogenes House-keeping Protein Secretion Systems

“…Integral membrane proteins are translocated cotranslationally. As soon as their nascent chain exits the ribosome, they are recognized by the signal recognition particle (SRP) 1 , a ribonucleoprotein complex composed of the GTPase Ffh, its newly identified modulator YlxM (32) and the SRP 4.5S RNA. The SRP guides the ribosome-nascent chain complexes to the SecA-SecYEG translocon via the transmembrane SRP receptor FtsY (33,34).…”