Flagellar Structures from the Bacterium Caulobacter crescentus and Implications for Phage ϕ CbK Predation of Multiflagellin Bacteria

Abstract

Caulobacter crescentus is a Gram-negative alphaproteobacterium that commonly lives in oligotrophic fresh- and saltwater environments. C. crescentus is a host to many bacteriophages, including ϕCbK and ϕCbK-like bacteriophages, which require interaction with the bacterial flagellum and pilus complexes during adsorption. It is commonly thought that the six paralogs of the flagellin gene present in C. crescentus are important for bacteriophage evasion. Here, we show that deletion of specific flagellins in C. crescentus can indeed attenuate ϕCbK adsorption efficiency, although no single deletion completely ablates ϕCbK adsorption. Thus, the bacteriophage ϕCbK likely recognizes a common motif among the six known flagellins in C. crescentus with various degrees of efficiency. Interestingly, we observe that most deletion strains still generate flagellar filaments, with the exception of a strain that contains only the most divergent flagellin, FljJ, or a strain that contains only FljN and FljO. To visualize the surface residues that are likely recognized by ϕCbK, we determined two high-resolution structures of the FljK filament, with and without an amino acid substitution that induces straightening of the filament. We observe posttranslational modifications on conserved surface threonine residues of FljK that are likely O-linked glycans. The possibility of interplay between these modifications and ϕCbK adsorption is discussed. We also determined the structure of a filament composed of a heterogeneous mixture of FljK and FljL, the final resolution of which was limited to approximately 4.6 Å. Altogether, this work builds a platform for future investigations of how phage ϕCbK infects C. crescentus at the molecular level. IMPORTANCE Bacterial flagellar filaments serve as an initial attachment point for many bacteriophages to bacteria. Some bacteria harbor numerous flagellin genes and are therefore able to generate flagellar filaments with complex compositions, which is thought to be important for evasion from bacteriophages. This study characterizes the importance of the six flagellin genes in C. crescentus for infection by bacteriophage ϕCbK. We find that filaments containing the FljK flagellin are the preferred substrate for bacteriophage ϕCbK. We also present a high-resolution structure of a flagellar filament containing only the FljK flagellin, which provides a platform for future studies on determining how bacteriophage ϕCbK attaches to flagellar filaments at the molecular level.

Structural basis of flagellar filament asymmetry and supercoil templating by Leptospira spirochete sheath proteins

“…In contrast to previously reported flagellar filament structures (Yonekura, Maki-Yonekura et al 2003, Maki-Yonekura, Yonekura et al 2010, Wang, Burrage et al 2017, Kreutzberger, Ewing et al 2020, Montemayor, Ploscariu et al 2021), which correspond to idealized straight helical assemblies, our fcpA - structure is curved and exhibits a pronounced supercoil. The shape parameters measured for our fcpA - structure (left-handed supercoil with pitch and diameter of 2.0 μm and 0.4 μm respectively) are distinctly different than those measured for the wild-type filament (pitch and diameter of 0.22 μm and 0.45 μm respectively) (Gibson, Trajtenberg et al 2020).…”

“…S1C), visualizing all residues of FlaB and allowing a complete atomic model to be built. As observed in other flagellar structures (Yonekura, Maki-Yonekura et al 2003, Maki-Yonekura, Yonekura et al 2010, Wang, Burrage et al 2017, Blum, Filippidou et al 2019, Kreutzberger, Ewing et al 2020, Montemayor, Ploscariu et al 2021, FlaB in our structure has two distinct subdomains (D0 and D1; Fig. 1D), each composed mainly of an extended bundle of alpha helices.…”

“…Leptospira also express at least two additional sheath proteins not found in other spirochetes: Flagellar Coiling Proteins FcpA and FcpB, which contribute to the coiled shape of wild-type filaments (Wunder, Figueira et al 2016, Wunder, Slamti et al 2018. As in other bacterial flagella (Wyss 1998, Kurniyati, Kelly et al 2017, Blum, Filippidou et al 2019, Kreutzberger, Ewing et al 2020, Montemayor, Ploscariu et al 2021, it is presumed that the Leptospira filament core is glycosylated (Holzapfel, Bonhomme et al 2020).…”

Identification of Receptor Binding Proteins in Flagellotropic Agrobacterium Phage 7-7-1

“…Initially, it was postulated that the tail fibers were responsible for mediating interactions between phage 7-7-1 and host flagella, but this hypothesis has not been tested [ 41 ]. Although a “nut and bolt” model for the translocation of bacteriophage χ and other flagella-dependent phages has been proposed, it becomes increasingly clear that there is no uniform mode of phage translocation along the flagellar filament [ 12 , 45 , 51 ]. This is not only a result of the differences in phage appendages (i.e., head versus tail fibers), but also due to the different modes of flagellar rotation and filament structure.…”

“…Phage 7-7-1 is a flagellotropic or flagella-dependent member of the Myoviridae family [ 23 , 41 , 42 ]. This class of phages begin infection by interacting and binding to the flagellar filaments of their bacterial hosts [ 12 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 ]. Flagella are used by bacteria to move throughout their environments and are powered by a motor located at the flagellar base that relies on proton motive force [ 48 ].…”