Dynamics of Bacterial Signal Recognition Particle at a Single Molecule Level

Abstract

We have studied the localization and dynamics of bacterial Ffh, part of the SRP complex, its receptor FtsY, and of ribosomes in the Gamma-proteobacterium Shewanella putrefaciens. Using structured illumination microscopy, we show that ribosomes show a pronounced accumulation at the cell poles, whereas SRP and FtsY are distributed at distinct sites along the cell membrane, but they are not accumulated at the poles. Single molecule dynamics can be explained by assuming that all three proteins/complexes move as three distinguishable mobility fractions: a low mobility/static fraction may be engaged in translation, medium-fast diffusing fractions may be transition states, and high mobility populations likely represent freely diffusing molecules/complexes. Diffusion constants suggest that SRP and FtsY move together with slow-mobile ribosomes. Inhibition of transcription leads to loss of static molecules and reduction of medium-mobile fractions, in favor of freely diffusing subunits, while inhibition of translation appears to stall the medium mobile fractions. Depletion of FtsY leads to aggregation of Ffh, but not to loss of the medium mobile fraction, indicating that Ffh/SRP can bind to ribosomes independently from FtsY. Heat maps visualizing the three distinct diffusive populations show that while static molecules are mostly clustered at the cell membrane, diffusive molecules are localized throughout the cytosol. The medium fast populations show an intermediate pattern of preferential localization, suggesting that SRP/FtsY/ribosome transition states may form within the cytosol to finally find a translocon.

Real-Time Messenger RNA Dynamics in Bacillus subtilis

“…Even given the noise generated by non-specifically binding MS2-mVenus protein, we found evidence for mRNA movement through the entire cell and, for confined motion, indicative of mRNA being within a larger structure such as bound by translating ribosomes. In agreement with the finding that free ribosomal subunits can move throughout the cells, while translating 70S ribosomes and polysomes are present at polar regions and at peripheral site surrounding the nucleoids (Sanamrad et al, 2014;Mayer et al, 2021), we found confined motion predominantly at sites excluded from the nucleoids, that is, the cell periphery and the cell poles. Although singlemolecule data could be best explained by assuming three distinct populations of molecules with different average diffusion constants, we cannot exclude that only statistically positioned mRNAs (engaged in translation) and freely mobile molecule exist.…”

“…Interestingly, observed diffusion constants for different mRNAs were rather similar. The populations with the highest DC, which we interpret to represent freely mobile molecules, showed a mobility much lower than that of even large proteins (Schibany et al, 2018;Mayer et al, 2021), in agreement with the large size of mRNAs. Interestingly, these molecules diffused even through the nucleoids that have been discussed to present diffusion barriers within bacterial cells.…”

“…The products of ypbS and ypzF are of presently unknown function. FtsY is the membrane-associated receptor for the signal recognition particle ( Braig et al, 2009 ) and localizes at the membrane, but also diffuses through the cell ( Mayer et al, 2021 ); RNase Y is also membrane-associated ( Hamouche et al, 2020 ), whereas SMC localizes to the nucleoids ( Schibany et al, 2018 ). The mreB-minD operon comprises genes for cell shape-determining proteins MreB (soluble), MreC, and MreD (membrane proteins), part of the Rod complex for lateral cell wall synthesis ( Dominguez-Escobar et al, 2011 ; Dersch et al, 2020 ).…”

“…The products of ypbS and ypzF are of presently unknown function. FtsY is the membrane-associated receptor for the signal recognition particle (Braig et al, 2009) and localizes at the membrane, but also diffuses through the cell (Mayer et al, 2021); RNase Y is also membrane-associated (Hamouche et al, 2020), whereas SMC localizes to the nucleoids 1 Artificial mRNAs with one MS2-binding site and 1,700-bp plasmid sequence (not added in the table).…”

Antibiotic Drug screening and Image Characterization Toolbox (A.D.I.C.T.): a robust imaging workflow to monitor antibiotic stress response in bacterial cells in vivo

“…Andor Solis as camera software using at least 2000 frames with integration times of 17.76 ms. Further information about the raw data used in this study is covered in Mayer et al 2021 15 .…”