Two novel heteropolymer-forming proteins maintain multicellular shape of the cyanobacteriumAnabaenasp. PCC 7120

Abstract

23The determinants of bacterial cell shape are extensively studied in unicellular forms. 24Nonetheless, the mechanisms that shape bacterial multicellular forms remain understudied. 25Here we study coiled-coil rich proteins (CCRPs) in the multicellular cyanobacterium Anabaena 26 sp. PCC 7120 (hereafter Anabaena). Our results reveal two CCPRs, termed LfiA and LfiB (for 27 linear filament), which assemble into a heteropolymer that traverses the longitudinal cell axis. 28Two additional CCRPs, CypS (for cyanobacterial polar scaffold) and CeaR (for cyanobacterial 29 elongasome activity regulator), form a polar proteinaceous scaffold and regulate MreB activity, 30 respectively. Deletion mutants of these CCRPs are characterized by impaired filament shape 31 and decreased viability. Our results indicate that the four CCRPs form a proteinaceous network 32 that stabilizes the Anabaena multicellular filament. We propose that this cytoskeletal network 33 is essential for the manifestation of the linear filament phenotype in Anabaena. 34 (a multi-enzyme complex), is a regulator of longitudinal PG biogenesis, and thus it plays a 49 crucial role in the adaption to different environments and prokaryotic multicellularity. 50The key hallmarks of permanent bacterial multicellularity are morphological 51 differentiation and a well-defined and reproducible shape, termed patterned multicellularity 3 . 52Unlike biofilms, patterned multicellular structures are the result of either coordinated swarming 53 or developmental aggregation behavior as in myxobacteria 9 . Additional factors include cell 54 division, proliferation and cell differentiation as in sporulating actinomycetes 10 and 55 cyanobacterial filaments 11,12 . In myxobacteria as well as in actinomycetes, it has been shown 56 that patterned multicellular traits are dependent on the coordinated function of different coiled-57 coil-rich proteins (CCRPs). Reminiscent of eukaryotic intermediate filaments (IFs) 13,14 , many 58 bacterial CCRPs were shown to perform analogous cytoskeletal functions through their ability 59 to self-assemble into distinct filaments in vitro and in vivo [15][16][17][18][19] . Unlike FtsZ or MreB 20,21 , 60 bacterial IF-like CCRPs do not require any additional co-factors for polymerization in vitro 22 . 61For example, in Myxococcus xanthus, the coordinated swarming and aggregation into fruiting 62 bodies is mediated by its gliding motility 23 , which strictly depends on the filament-forming 63 CCRP AglZ 24 . AglZ is organized in a large multi-protein complex that governs gliding motility 64 in synergy with MreB, which still retained its PG synthesis function but was also co-opted for 65 gliding motility in M. xanthus [25][26][27][28] . Actinobacteria, such as Streptomyces species, grow by 66 building new cell wall (i.e. PG) only at the cell poles, independent of MreB 29,30 , which is 67 strikingly different from how most other bacteria grow 31 . This characteristic polar growth mode 68 is organized by a cytoskeletal network of at least three CCRPs -DivIVA...