Summary Leptospira are unique among bacteria based on their helical cell morphology with hook-shaped ends and the presence of periplasmic flagella (PF) with pronounced spontaneous supercoiling. The factors that provoke such supercoiling, as well as the role that PF coiling plays in generating the characteristic hook-end cell morphology and motility, have not been elucidated. We have now identified an abundant protein from the pathogen L. interrogans, exposed on the PF surface, and named it Flagellar-coiling protein A (FcpA). The gene encoding FcpA is highly conserved among Leptospira and was not found in other bacteria. fcpA- mutants, obtained from clinical isolates or by allelic exchange, had relatively straight, smaller-diameter PF, and were not able to produce translational motility. These mutants lost their ability to cause disease in the standard hamster model of leptospirosis. Complementation of fcpA restored the wild-type morphology, motility and virulence phenotypes. In summary, we identified a novel Leptospira 36-kDa protein, the main component of the spirochete's PF sheath, and a key determinant of the flagella's coiled structure. FcpA is essential for bacterial translational motility and to enable the spirochete to penetrate the host, traverse tissue barriers, disseminate to cause systemic infection, and reach target organs.

T he bacterial flagellum is a sophisticated macromolecular complex. Its structure and assembly have been well studied in two model organisms, Escherichia coli and Salmonella enterica serovar Typhimurium (for reviews, see references 5, 13, 36, and 58). The flagellum is composed of at least 25 different proteins that can be grouped into three physical parts: the basal body, the flagellar hook, and the filament. The basal body is imbedded within the cell envelope and works as a reversible rotary motor; the flagellar hook and filament extend outwards to the cell exterior and function as a universal joint and a propeller, respectively. The basal body is very complex and consists of several functional units: the membranesupramembrane (MS)-C ring (rotor), the rod (driveshaft), the L-P rings (bushings), the stator (torque generator), and the flagellar export apparatus. The motor is driven by an inward-directed electrochemical gradient of protons or sodium. The torque generated by the motor is mechanically transmitted to the filament via the rod-hook complex, leading to the rotation of flagellar filament, which propels the bacterial cells forward.Flagellar assembly is a sequential process (for reviews, see references 1 and 13). It begins with the MS ring assembly. Built onto the MS ring is a hollow rod that spans the periplasmic space. After formation of the MS ring/rod complex, the FlgI and FlgH proteins assemble around the rod to form the P and L rings, respectively. The hook and filament proteins are subsequently assembled on the rod. The flagellar rod begins with the MS ring and stops at the flagellar hook. Thus, it needs to penetrate the peptidoglycan (PG) layer during flagellar formation. It has been postulated that FlgJ is essential for flagellar rod formation (25,45), with the N-terminal domain (rod-capping) acting as a scaffold for rod assembly and the C-terminal domain functioning as a PG hydrolase (PGase), which makes a hole in the PG layer to allow rod penetration. In S. Typhimurium, flgJ null mutants are aflagellated and nonmotile, while mutants that do not express the PGase domain produce fewer flagella and show poor motility (25, 45). However, the PGase domain is absent in the FlgJ homologs from several bacterial phyla, including Alphaproteobacteria, Deltaproteobacteria, and Epsilonproteobacteria and Spirochaetes (44). As there is only one domain, these homologs are referred to as "single-domain FlgJ." The function of these FlgJ homologs remains unknown.Spirochetes are a group of motile bacteria that have a unique morphology and are able to swim in highly viscous gel-like environments (for reviews, see references 11 and 31). It is believed that motility plays a critical role in the biology of spirochetes and in the processes of diseases caused by pathogenic spirochetes (9,11,16,32,55). Spirochetes swim by means of two rotating bundles of periplasmic flagella (PFs) that reside between the outer membrane and cell cylinder (23,32,38,49). PFs are structurally similar to the flagella of other bacteria, as each ...

CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against genetic invaders, such as bacteriophages. The systems integrate short sequences from the phage genome into the bacterial CRISPR array. These ‘spacers’ provide sequence-specific immunity but drive natural selection of evolved phage mutants that escape the CRISPR-Cas defence. Spacer acquisition occurs by either naive or primed adaptation. Naive adaptation typically results in the incorporation of a single spacer. By contrast, priming is a positive feedback loop that often results in acquisition of multiple spacers, which occurs when a pre-existing spacer matches the invading phage. We predicted that single and multiple spacers, representative of naive and primed adaptation, respectively, would cause differing outcomes after phage infection. We investigated the response of two phages, ϕTE and ϕM1, to the Pectobacterium atrosepticum type I-F CRISPR-Cas system and observed that escape from single spacers typically occurred via point mutations. Alternatively, phages escaped multiple spacers through deletions, which can occur in genes encoding structural proteins. Cryo-EM analysis of the ϕTE structure revealed shortened tails in escape mutants with tape measure protein deletions. We conclude that CRISPR-Cas systems can drive phage genetic diversity, altering morphology and fitness, through selective pressures arising from naive and primed acquisition events. This article is part of a discussion meeting issue ‘The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems’.

Pseudomonas syringae pv. actinidiae is an economically significant pathogen responsible for severe bacterial canker of kiwifruit (Actinidia sp.). Bacteriophages infecting this phytopathogen have potential as biocontrol agents as part of an integrated approach to the management of bacterial canker, and for use as molecular tools to study this bacterium. A variety of bacteriophages were previously isolated that infect P. syringae pv. actinidiae, and their basic properties were characterized to provide a framework for formulation of these phages as biocontrol agents. Here, we have examined in more detail φPsa17, a phage with the capacity to infect a broad range of P. syringae pv. actinidiae strains and the only member of the Podoviridae in this collection. Particle morphology was visualized using cryo-electron microscopy, the genome was sequenced, and its structural proteins were analysed using shotgun proteomics. These studies demonstrated that φPsa17 has a 40,525 bp genome, is a member of the T7likevirus genus and is closely related to the pseudomonad phages φPSA2 and gh-1. Eleven structural proteins (one scaffolding) were detected by proteomics and φPsa17 has a capsid of approximately 60 nm in diameter. No genes indicative of a lysogenic lifecycle were identified, suggesting the phage is obligately lytic. These features indicate that φPsa17 may be suitable for formulation as a biocontrol agent of P. syringae pv. actinidiae.

Local translation of membrane proteins in neuronal subcellular domains like soma, dendrites and axon termini is well-documented. In this study, we isolated the electrical signaling unit of an axon by dissecting giant axons from mature squids (Dosidicus gigas). Axoplasm extracted from these axons was found to contain ribosomal RNAs, ~8000 messenger RNA species, many encoding the translation machinery, membrane proteins, translocon and signal recognition particle (SRP) subunits, endomembrane-associated proteins, and unprecedented proportions of SRP RNA (~68% identical to human homolog). While these components support endoplasmic reticulum-dependent protein synthesis, functional assessment of a newly synthesized membrane protein in axolemma of an isolated axon is technically challenging. Ion channels are ideal proteins for this purpose because their functional dynamics can be directly evaluated by applying voltage clamp across the axon membrane. We delivered in vitro transcribed RNA encoding native or Drosophila voltage-activated Shaker KV channel into excised squid giant axons. We found that total K+ currents increased in both cases; with added inactivation kinetics on those axons injected with RNA encoding the Shaker channel. These results provide unambiguous evidence that isolated axons can exhibit de novo synthesis, assembly and membrane incorporation of fully functional oligomeric membrane proteins.