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Abstract A contractile sheath and rigid tube assembly is a widespread apparatus used by bacteriophages, tailocins, and the bacterial type VI secretion system to penetrate cell membranes. In this mechanism, contraction of an external sheath powers the motion of an inner tube through the membrane. The structure, energetics, and mechanism of the machinery imply rigidity and straightness. The contractile tail ofAgrobacterium tumefaciensbacteriophage Milano is flexible and bent to varying degrees, which sets it apart from other contractile tail-like systems. Here, we report structures of the Milano tail including the sheath-tube complex, baseplate, and putative receptor-binding proteins. The flexible-to-rigid transformation of the Milano tail upon contraction can be explained by unique electrostatic properties of the tail tube and sheath. All components of the Milano tail, including sheath subunits, are crosslinked by disulfides, some of which must be reduced for contraction to occur. The putative receptor-binding complex of Milano contains a tailspike, a tail fiber, and at least two small proteins that form a garland around the distal ends of the tailspikes and tail fibers. Despite being flagellotropic, Milano lacks thread-like tail filaments that can wrap around the flagellum, and is thus likely to employ a different binding mechanism. 

Abstract Large gaps exist in our understanding of how bacteriophages, the most abundant biological entities on Earth, assemble and function. The structure of the “neck” region, where the DNA-filled capsid is connected to the host-recognizing tail remains poorly understood. We describe cryo-EM structures of the neck, the neck-capsid and neck-tail junctions, and capsid of theAgrobacteriumphage Milano. The Milano neck 1 protein connects the 12-fold symmetrical neck to a 5-fold vertex of the icosahedral capsid. Comparison of Milano neck 1 homologs leads to four proposed classes, likely evolved from the simplest one in siphophages to more complex ones in myo- and podophages. Milano neck is surrounded by the atypical collar, which covalently crosslinks the tail sheath to neck 1. The Milano capsid is decorated with three types of proteins, a minor capsid protein (mCP) and two linking proteins crosslinking the mCP to the major capsid protein. The extensive network of disulfide bonds within and between neck, collar, capsid and tail provides an exceptional structural stability to Milano. 

The bacterial flagellum is a rotary motor organelle and important virulence factor that propels motile pathogenic bacteria, such asSalmonella enterica, through their surroundings. Bacteriophages, or phages, are viruses that solely infect bacteria. As such, phages have myriad applications in the healthcare field, including phage therapy against antibiotic-resistant bacterial pathogens. Bacteriophage χ (Chi) is a flagellum-dependent (flagellotropic) bacteriophage, which begins its infection cycle by attaching its long tail fiber to theS.entericaflagellar filament as its primary receptor. The interactions between phage and flagellum are poorly understood, as are the reasons that χ only kills certainSalmonellaserotypes while others entirely evade phage infection. In this study, we used molecular cloning, targeted mutagenesis, heterologous flagellin expression, and phage-host interaction assays to determine which domains within the flagellar filament protein flagellin mediate this complex interaction. We identified the antigenic N- and C-terminal D2 domains as essential for phage χ binding, with the hypervariable central D3 domain playing a less crucial role. Here, we report that the primary structure of theSalmonellaflagellin D2 domains is the major determinant of χ adhesion. The phage susceptibility of a strain is directly tied to these domains. We additionally uncovered important information about flagellar function. The central and most variable domain, D3, is not required for motility inS. Typhimurium 14028s, as it can be deleted or its sequence composition can be significantly altered with minimal impacts on motility. Further knowledge about the complex interactions between flagellotropic phage χ and its primary bacterial receptor may allow genetic engineering of its host range for use as targeted antimicrobial therapy against motile pathogens of the χ-host generaSalmonella,Escherichia, orSerratia. 

Abstract Flagellar filaments function as the propellers of the bacterial flagellum and their supercoiling is key to motility. The outer domains on the surface of the filament are non-critical for motility in many bacteria and their structures and functions are not conserved. Here, we show the atomic cryo-electron microscopy structures for flagellar filaments from enterohemorrhagic Escherichia coli O157:H7, enteropathogenic E. coli O127:H6, Achromobacter , and Sinorhizobium meliloti , where the outer domains dimerize or tetramerize to form either a sheath or a screw-like surface. These dimers are formed by 180° rotations of half of the outer domains. The outer domain sheath (ODS) plays a role in bacterial motility by stabilizing an intermediate waveform and prolonging the tumbling of E. coli cells. Bacteria with these ODS and screw-like flagellar filaments are commonly found in soil and human intestinal environments of relatively high viscosity suggesting a role for the dimerization in these environments. 

Bacteriophages (phages) are the most abundant biological entities in the biosphere. As viruses that solely infect bacteria, phages have myriad healthcare and agricultural applications including phage therapy and antibacterial treatments in the foodservice industry. Phage therapy has been explored since the turn of the twentieth century but was no longer prioritized following the invention of antibiotics. As we approach a post-antibiotic society, phage therapy research has experienced a significant resurgence for the use of phages against antibiotic-resistant bacteria, a growing concern in modern medicine. Phages are extraordinarily diverse, as are their host receptor targets. Flagellotropic (flagellum-dependent) phages begin their infection cycle by attaching to the flagellum of their motile host, although the later stages of the infection process of most of these phages remain elusive. Flagella are helical appendages required for swimming and swarming motility and are also of great importance for virulence in many pathogenic bacteria of clinical relevance. Not only is bacterial motility itself frequently important for virulence, as it allows pathogenic bacteria to move toward their host and find nutrients more effectively, but flagella can also serve additional functions including mediating bacterial adhesion to surfaces. Flagella are also a potent antigen recognized by the human immune system. Phages utilizing the flagellum for infections are of particular interest due to the unique evolutionary tradeoff they force upon their hosts: by downregulating or abolishing motility to escape infection by a flagellotropic phage, a pathogenic bacterium would also likely attenuate its virulence. This factor may lead to flagellotropic phages becoming especially potent antibacterial agents. This review outlines past, present, and future research of flagellotropic phages, including their molecular mechanisms of infection and potential future applications. 

The rapid discovery of new and diverse bacteriophages has driven the innovation of approaches aimed at detailing interactions with their bacterial hosts. Previous studies on receptor binding proteins (RBPs) mainly relied on their identification in silico and are based on similarities to well-characterized systems. Thus, novel phage RBPs unlike those currently annotated in genomic and proteomic databases remain largely undiscovered. In this study, we employed a screen to identify RBPs in flagellotropic Agrobacterium phage 7-7-1. Flagellotropic phages utilize bacterial flagella as receptors. The screen identified three candidate RBPs, Gp4, Gp102, and Gp44. Homology modelling predicted that Gp4 is a trimeric, tail associated protein with a central β-barrel, while the structure and function of Gp102 and Gp44 are less obvious. Studies with purified Gp41-247 confirmed its ability to bind and interact with host cells, highlighting the robustness of the RBP screen. We also discovered that Gp41-247 inhibits the growth of host cells in a motility and lipopolysaccharide (LPS) dependent fashion. Hence, our results suggest interactions between Gp41-247, rotating flagellar filaments and host glycans to inhibit host cell growth, which presents an impactful and intriguing focus for future studies. 

Antimicrobial resistance is a large concern in the health care field. With more multidrug-resistant bacterial pathogens emerging, other techniques for eliminating bacterial infections are being explored.