Phosphorylation of a Novel Cytoskeletal Protein (RsmP) Regulates Rod-shaped Morphology in Corynebacterium glutamicum

Abstract

Corynebacteria grow by wall extension at the cell poles, with DivIVA being an essential protein orchestrating cell elongation and morphogenesis. DivIVA is considered a scaffolding protein able to recruit other proteins and enzymes involved in polar peptidoglycan biosynthesis. Partial depletion of DivIVA induced overexpression of cg3264, a previously uncharacterized gene that encodes a novel coiled coil-rich protein specific for corynebacteria and a few other actinomycetes. By partial depletion and overexpression of Cg3264, we demonstrated that this protein is an essential cytoskeletal element needed for maintenance of the rod-shaped morphology of Corynebacterium glutamicum, and it was therefore renamed RsmP (rod-shaped morphology protein). RsmP forms long polymers in vitro in the absence of any cofactors, thus resembling eukaryotic intermediate filaments. We also investigated whether RsmP could be regulated post-translationally by phosphorylation, like eukaryotic intermediate filaments. RsmP was phosphorylated in vitro by the PknA protein kinase and to a lesser extent by PknL. A mass spectrometric analysis indicated that phosphorylation exclusively occurred on a serine (Ser-6) and two threonine (Thr-168 and Thr-211) residues. We confirmed that mutagenesis to alanine (phosphoablative protein) totally abolished PknA-dependent phosphorylation of RsmP. Interestingly, when the three residues were converted to aspartic acid, the phosphomimetic protein accumulated at the cell poles instead of making filaments along the cell, as observed for the native or phosphoablative RsmP proteins, indicating that phosphorylation of RsmP is necessary for directing cell growth at the cell poles.

The Ser/Thr protein kinase AfsK regulates polar growth and hyphal branching in the filamentous bacteria Streptomyces

“…Such a role for STKs in controlling assemblages of coiled-coil proteins is well known in eukaryotes, a classic example being the disassembly of the nuclear lamina mediated by cyclin-dependent kinases (41). In addition, it was recently reported that the assembly and subcellular localization of the coiled-coil protein RsmP in Corynebacterium glutamicum is affected by phosphorylation (42). However, it cannot be excluded that phosphorylation may also influence other aspects of DivIVA behavior, such as its interaction with other proteins or the membrane.…”

Nucleotide‐independent cytoskeletal scaffolds in bacteria

“…(B) Domain structure of bacterial CCRPs. The positions of coiled‐coil regions in crescentin, FilP, and RsmP are shown as reported previously [Ausmees et al, ; Fiuza et al, ; Walshaw et al, ]. Coiled‐coil segments in Ccrp59 and Ccrp1143 were determined using the COILS server [Lupas et al, ].…”

“…B) from the rod‐shaped organism Corynebacterium glutamicum . RsmP is essential for viability, and its depletion leads to rounding up of the cells [Fiuza et al, ]. Consistent with its ability to polymerize spontaneously in vitro, RsmP assembles into massive, long filaments upon overproduction in C. glutamicum .…”

Polarity and the diversity of growth mechanisms in bacteria

“…DivIVA depletion leads to upregulation of RsmP, which has similarity to eukaryotic intermediate filaments [29]. RsmP is an essential protein and its homologs are unique to the Actinobacteria.…”