Functional Genomics Uncovers Pleiotropic Role of Rhomboids in Corynebacterium glutamicum.

Ansgar Poetsch,Andrea Luenenschloss,Frank Ter Veld,Tobias M Neddermann,Stefan P Albaum,Volker F Wendisch

doi:10.3389/fmicb.2022.771968

Abstract

The physiological role of ubiquitous rhomboid proteases, membrane-integral proteins that cleave their substrates inside the lipid bilayer, is still ill-defined in many prokaryotes. The two rhomboid genes cg0049 and cg2767 of Corynebacterium glutamicum were mutated and it was the aim of this study to investigate consequences in respect to growth phenotype, stress resistance, transcriptome, proteome, and lipidome composition. Albeit increased amount of Cg2767 upon heat stress, its absence did not change the growth behavior of C. glutamicum during exponential and stationary phase. Quantitative shotgun mass spectrometry was used to compare the rhomboid mutant with wild type strain and revealed that proteins covering diverse cellular functions were differentially abundant with more proteins affected in the stationary than in the exponential growth phase. An observation common to both growth phases was a decrease in ribosomal subunits and RNA polymerase, differences in iron uptake proteins, and abundance changes in lipid and mycolic acid biosynthesis enzymes that suggested a functional link of rhomboids to cell envelope lipid biosynthesis. The latter was substantiated by shotgun lipidomics in the stationary growth phase, where in a strain-dependent manner phosphatidylglycerol, phosphatidic acid, diacylglycerol and phosphatidylinositol increased irrespective of cultivation temperature.

Highlights

In their natural habitat, bacteria must constantly adapt to changing environmental conditions, such as a sudden increase in temperature
Rhomboids comprise the largest family of all the intramembrane proteases (Weihofen and Martoglio, 2003; Urban and Dickey, 2011); genome sequence analysis revealed their almost ubiquitous occurrence in archaea, bacteria, and eukaryotes, though protein sequence homologies can be as low as 6% (Lemberg and Freeman, 2007)
Cleavage of its N-terminal domain by the rhomboid AarA activates TatA for quorum signal liberation. This N-terminal domain is absent in the vast majority of bacterial A subunits of the Tat exporter, TatA processing does not provide a hallmark on prokaryotic rhomboid substrates and function

Summary

Introduction

Bacteria must constantly adapt to changing environmental conditions, such as a sudden increase in temperature. Rhomboids comprise the largest family of all the intramembrane proteases (Weihofen and Martoglio, 2003; Urban and Dickey, 2011); genome sequence analysis revealed their almost ubiquitous occurrence in archaea, bacteria, and eukaryotes, though protein sequence homologies can be as low as 6% (Lemberg and Freeman, 2007). Rhomboids are a rare case of membrane proteins, where structure and mechanism have been explored at an advanced level, whereas our knowledge on physiological function and natural substrates is falling short for most bacteria. Cleavage of its N-terminal domain by the rhomboid AarA activates TatA for quorum signal liberation. This N-terminal domain is absent in the vast majority of bacterial A subunits of the Tat exporter, TatA processing does not provide a hallmark on prokaryotic rhomboid substrates and function. For Shigella sonnei involvement of rhomboids GlpG and Rhom in membrane protein quality control with HybA, FdnH, and FdoH as substrates was demonstrated (Liu et al, 2020)

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