Identification and characterization of novel filament-forming proteins in cyanobacteria

Abstract

Filament-forming proteins in bacteria function in stabilization and localization of proteinaceous complexes and replicons; hence they are instrumental for myriad cellular processes such as cell division and growth. Here we present two novel filament-forming proteins in cyanobacteria. Surveying cyanobacterial genomes for coiled-coil-rich proteins (CCRPs) that are predicted as putative filament-forming proteins, we observed a higher proportion of CCRPs in filamentous cyanobacteria in comparison to unicellular cyanobacteria. Using our predictions, we identified nine protein families with putative intermediate filament (IF) properties. Polymerization assays revealed four proteins that formed polymers in vitro and three proteins that formed polymers in vivo. Fm7001 from Fischerella muscicola PCC 7414 polymerized in vitro and formed filaments in vivo in several organisms. Additionally, we identified a tetratricopeptide repeat protein-All4981-in Anabaena sp. PCC 7120 that polymerized into filaments in vitro and in vivo. All4981 interacts with known cytoskeletal proteins and is indispensable for Anabaena viability. Although it did not form filaments in vitro, Syc2039 from Synechococcus elongatus PCC 7942 assembled into filaments in vivo and a Δsyc2039 mutant was characterized by an impaired cytokinesis. Our results expand the repertoire of known prokaryotic filament-forming CCRPs and demonstrate that cyanobacterial CCRPs are involved in cell morphology, motility, cytokinesis and colony integrity. Species in the phylum Cyanobacteria present a wide morphological diversity, ranging from unicellular to multicellular organisms. Unicellular cyanobacteria of the Synechocystis and Synechococcus genera are characterized by a round or rod-shaped morphology, respectively, and many strains are motile. Species of the Nostocales order are multicellular and differentiate three types of specialized cells including heterocysts, which fix atmospheric nitrogen under aerobic conditions, hormogonia that are reproductive motile filaments and akinetes, which are dormant cells that are resistant to desiccation. Within the Nostocales, species of the Nostocaceae (e.g., Anabaena, Nostoc) form linear trichomes, while cells in the Hapalosiphonaceae and Chlorogloepsidaceae divide in more than one plane to form true-branching trichomes as in Fischerella or multiseriate trichomes (more than one filament in a row) as in Chlorogloeopsis 1. Notably, cells within a single trichome of a multicellular cyanobacterium can differ in size, form or cell wall composition, which may be attributed to different stages of cell differentiation (or phenotypic heterogeneity) and varying environmental cues 2,3. Cells in the Anabaena sp. PCC 7120 (hereafter Anabaena) trichome are linked by a shared peptidoglycan sheet and an outer membrane 4. Anabaena cells communicate and exchange nutrients through intercellular cell-cell connections, called septal junctions, which are thought to comprise the septal junction proteins SepJ, FraC and FraD 5,6. SepJ is essential for the mult...

Structural Determinants and Their Role in Cyanobacterial Morphogenesis

“…Using a streamlined approach to readily test several candidate CCRPs with a newly developed in vitro polymerization assay allowed us to detect four novel filament-forming CCRPs in cyanobacteria. In Synechocystis , Slr1301 (termed HmpF Syn ) is a homologous protein to HmpF from Nostoc punctiforme [ 206 ], which, similar to its homolog, was found to be involved in Synechocystis twitching motility (as also previously identified by [ 207 ]), possibly through its interaction with the pilus ATPase PilB [ 204 ]. Despite its high CC content, HmpF Syn did not assemble into IF-like polymers in vitro and in vivo, highlighting that the pure presence of many CC domains is not sufficient to predict IF-like properties.…”

“…Notably, no S-layer has been detected in Anabaena [ 209 ], and a deletion of all4981 could not be obtained, hampering a functional dissection of All4981. In our study [ 204 ], we further found two Synechococcus CCRPs of which Synpcc7942_1139 (HmpF Syc ), a Synechococcus homolog to HmpF, is essential and has a severe impact on colony morphology, a novel trait of prokaryotic CCRPs. This essential property is in contrast to the non-essentiality of HmpF and HmpF Syn , suggesting specific functional adaptations to Synechococcus .…”

“…Given their seemingly ubiquitous involvement in cell shape, we recently searched for cyanobacterial CCRPs [ 204 ] that could be functionally involved in the manifestation of the enormous morphological diversity in the Cyanobacteria phylum [ 19 ]. In this study, we found that CCRPs are more prevalent in multicellular filamentous cyanobacteria compared to unicellular species.…”

“…In contrast to HmpF Syn , Slr7083 assembles into a honeycomb-like web of protein filaments in vitro and localizes circumferentially to the cell envelope. Slr7083 also affected the cellular motility of Synechocystis (although to a lesser extent) and as it directly interacted with HmpF Syn , both CCRPs could be involved in cellular motility [ 204 ], reminiscent of the H. pylori CCRPs that regulate swimming motility [ 202 ] and AglZ, which is involved in gliding motility [ 201 ]. We also showed that a protein specific to multicellular cyanobacteria, Fm7001, polymerizes into extremely stable filamentous sheets at 4.5 M urea, a concentration where the eukaryotic IF protein vimentin only exist as tetramers [ 208 ].…”

“…We also showed that a protein specific to multicellular cyanobacteria, Fm7001, polymerizes into extremely stable filamentous sheets at 4.5 M urea, a concentration where the eukaryotic IF protein vimentin only exist as tetramers [ 208 ]. This incredibly strong self-association capacity could function in the manifestation and stabilization of the F. muscicola trichome phenotype [ 204 ]. Interestingly, we also showed for the first time that a bacterial tetratricopeptide repeat (TPR) protein, All4981 from Anabaena , assembles into filamentous structures in vivo and in vitro, while interacting with a number of S-layer proteins.…”