Summary Pili are proteinaceous polymers of linked pilins that protrude from the cell surface of many bacteria and often mediate adherence and virulence. We investigated a set of 20 Bacteroidia pilins from the human microbiome whose structures and mechanism of assembly were unknown. Crystal structures and biochemical data revealed a diverse protein superfamily with a common Greek-key β-sandwich fold with two transthyretin-like repeats that polymerize into a pilus through a strand-exchange mechanism. The assembly mechanism of the central, structural pilins involves proteinase-assisted removal of their N-terminal β-strand, creating an extended hydrophobic groove that binds the C-terminal donor strands of the incoming pilin. Accessory pilins at the tip and base have unique structural features specific to their location, allowing initiation or termination of the assembly. The bacteroidia pilus therefore has a biogenesis mechanism that is distinct from other known pili and likely represents a different type of bacterial pilus.

Borrelia burgdorferi is a flat-wave, motile spirochete that causes Lyme disease. Motility is provided by periplasmic flagella (PFs) located between the cell cylinder and an outer membrane sheath. The structure of these PFs, which are composed of a basal body, a hook, and a filament, is similar to the structure of flagella of other bacteria. To determine if hook formation influences flagellin gene transcription in B. burgdorferi, we inactivated the hook structural gene flgE by targeted mutagenesis. In many bacteria, completion of the hook structure serves as a checkpoint for transcriptional control of flagellum synthesis and other chemotaxis and motility genes. Specifically, the hook allows secretion of the anti-sigma factor FlgM and concomitant late gene transcription promoted by 28 . However, the control of B. burgdorferi PF synthesis differs from the control of flagellum synthesis in other bacteria; the gene encoding 28 is not present in the genome of B. burgdorferi, nor are any 28 promoter recognition sequences associated with the motility genes. We found that B. burgdorferi flgE mutants lacked PFs, were rod shaped, and were nonmotile, which substantiates previous evidence that PFs are involved in both cell morphology and motility. Although most motility and chemotaxis gene products accumulated at wild-type levels in the absence of FlgE, mutant cells had markedly decreased levels of the flagellar filament proteins FlaA and FlaB. Further analyses showed that the reduction in the levels of flagellin proteins in the spirochetes lacking FlgE was mediated at the posttranscriptional level. Taken together, our results indicate that in B. burgdorferi, the completion of the hook does not serve as a checkpoint for transcriptional regulation of flagellum synthesis. In addition, we also present evidence that the hook protein in B. burgdorferi forms a high-molecular-weight complex and that formation of this complex occurs in the periplasmic space.Borrelia burgdorferi, the causative agent of Lyme disease, is a spirochete with a characteristic flat-wave morphology (24, 25; for a review, see reference 12). As a result of their unusual morphology, B. burgdorferi and other spirochete species have a highly specialized ability that allows them to traverse viscous gellike media (8). This unique swimming ability enables these bacteria to penetrate into specific host connective tissues and ecological niches (43). The importance of motility as a virulence factor has been implicated in several spirochete species, including Treponema denticola (42), Brachyspira hyodysenteriae (54), Borrelia garinii (60), and B. burgdorferi (10, 56).Motility in B. burgdorferi is provided by bundles of between 7 and 11 periplasmic flagella (PFs) that are subterminally attached near the cell ends. These PFs extend inward along the cell cylinder beneath an outer membrane sheath (6,24,28,46). B. burgdorferi PFs have a structure similar to that of flagella of other bacteria; a PF is composed of a basal body, a hook, and a filament containing a single major ...

SummaryAs part of our attempt to map the impact of acetyl phosphate (acetyl~P) on the entire network of twocomponent signal transduction pathways in Escherichia coli, we asked whether the influence of acetyl~P on capsular biosynthesis and flagellar biogenesis depends on the Rcs phosphorelay. To do so, we performed a series of epistasis experiments: mutations in the components of the pathway that controls acetyl~P levels were combined with mutations in components of the Rcs phosphorelay. Cells that did not synthesize acetyl~P produced no capsule under normally permissive conditions, while those that accumulated acetyl~P synthesized capsule under conditions previously considered to be nonpermissive. Acetyl~P-dependent capsular biosynthesis required both RcsB and RcsA, while the lack of RcsC restored capsular biosynthesis to acetyl~P-deficient cells. Similarly, acetyl~P-sensitive repression of flagellar biogenesis was suppressed by the loss of RcsB (but not of RcsA), while it was enhanced by the lack of RcsC. Taken together, these results show that both acetyl~P-sensitive activation of capsular biosynthesis and acetyl~P-sensitive repression of flagellar biogenesis require the Rcs phosphorelay. Moreover, they provide strong genetic support for the hypothesis that RcsC can function as either a kinase or a phosphatase dependent on environmental conditions. Finally, we learned that RcsB and RcsC inversely regulated the timing of flagellar biogenesis: rcsB mutants elaborated flagella prematurely, while rcsC mutants delayed their display of flagella. Temporal control of flagella biogenesis implicates the Rcs phosphorelay (and, by extension, acetyl~P) in the transition of motile, planktonic individuals into sessile biofilm communities.

SummaryThe mechanism of length control of the flagellar hook is under debate between two theories. One claims that the FliK directly measures the hook length as a molecular ruler, while the other claims that the cytoplasmic substructure measures the amount of hook subunits to determine the hook length. Both agree that the FliK C-terminal domain catalyses the substrate-specificity switch to terminate hook elongation. In this study, we systematically created fliK mutants with deletions and insertions at various sites within the FliK N-terminal domain and analysed their effects on the final hook length. Insertions of peptide fragments from the Yersinia YscP into FliK gave rise to hooks with defined lengths, which was proportional to the molecular size of the FliK-YscP chimeras. Among fliK deletion mutants, only those with small truncations in three specific sites of FliK produced hooks of a defined, shortened length. For the majority of deletion mutants, FliK was secreted, but hook length was not controlled. On the other hand, for some deletion mutants FliK was not secreted, but the hook length was controlled, indicating that FliK secretion is not necessary for hooklength control. We conclude that FliK regulates hook length as an internal molecular ruler.

Legume plants tightly control the development and number of symbiotic root nodules. In Lotus japonicus, this regulation requires HAR1 (a CLAVATA1-like receptor kinase) in the shoots, suggesting that a long-distance communication between the shoots and the roots may exist. To better understand its molecular basis, we isolated and characterized a novel hypernodulating mutant of L. japonicus named too much love (tml). Compared with the wild type, tml mutants produced much more nodules which densely covered a wider range of the roots. Reciprocal grafting showed that tml hypernodulation is determined by the root genotype. Moreover, grafting a har1 shoot onto a tml rootstock did not exhibit any obvious additive effects on the nodule number, which was further supported by double mutational analysis. These observations indicate that a shoot factor HAR1 and a root factor TML participate in the same genetic pathway which governs the long-distance signaling of nodule number control. We also showed that the inhibitory effect of TML on nodulation is likely to be local. Therefore, TML may function downstream of HAR1 and the gene product TML might serve as a receptor or mediator of unknown mobile signal molecules that are transported from the shoots to the roots.

We report the complete genome sequence of the deep-sea ␥-proteobacterium, Idiomarina loihiensis, isolated recently from a hydrothermal vent at 1,300-m depth on the Lo ihi submarine volcano, Hawaii. The I. loihiensis genome comprises a single chromosome of 2,839,318 base pairs, encoding 2,640 proteins, four rRNA operons, and 56 tRNA genes. A comparison of I. loihiensis to the genomes of other ␥-proteobacteria reveals abundance of amino acid transport and degradation enzymes, but a loss of sugar transport systems and certain enzymes of sugar metabolism. This finding suggests that I. loihiensis relies primarily on amino acid catabolism, rather than on sugar fermentation, for carbon and energy. Enzymes for biosynthesis of purines, pyrimidines, the majority of amino acids, and coenzymes are encoded in the genome, but biosynthetic pathways for Leu, Ile, Val, Thr, and Met are incomplete. Auxotrophy for Val and Thr was confirmed by in vivo experiments. The I. loihiensis genome contains a cluster of 32 genes encoding enzymes for exopolysaccharide and capsular polysaccharide synthesis. It also encodes diverse peptidases, a variety of peptide and amino acid uptake systems, and versatile signal transduction machinery. We propose that the source of amino acids for I. loihiensis growth are the proteinaceous particles present in the deep sea hydrothermal vent waters. I. loihiensis would colonize these particles by using the secreted exopolysaccharide, digest these proteins, and metabolize the resulting peptides and amino acids. In summary, the I. loihiensis genome reveals an integrated mechanism of metabolic adaptation to the constantly changing deep-sea hydrothermal ecosystem.hydrothermal vent

Motility often plays a decisive role in the survival of species. Five systems of motility have been studied in depth: those propelled by bacterial flagella, eukaryotic actin polymerization and the eukaryotic motor proteins myosin, kinesin and dynein. However, many organisms exhibit surprisingly diverse motilities, and advances in genomics, molecular biology and imaging have showed that those motilities have inherently independent mechanisms. This makes defining the breadth of motility nontrivial, because novel motilities may be driven by unknown mechanisms. Here, we classify the known motilities based on the unique classes of movement-producing protein architectures.Based on this criterion, the current total of independent motility systems stands at 18 types. In this perspective, we discuss these modes of motility relative to the latest phylogenetic Tree of Life and propose a history of motility. During the ~4 billion years since the emergence of life, motility arose in Bacteria with flagella and pili, and in Archaea with archaella. Newer modes of motility became possible in Eukarya with changes to the cell envelope. Presence or absence of a peptidoglycan layer, the acquisition of robust membrane dynamics, the enlargement of cells and environmental opportunities likely provided the context for the (co)evolution of novel types of motility. K E Y W O R D S appendage, cytoskeleton, flagella, membrane remodeling, Mollicutes, motor protein, peptidoglycan, three domains | 9Genes to Cells MIYATA eT Al.

SUMMARYEndosymbiotic infection of legume plants by Rhizobium bacteria is initiated through infection threads (ITs) which are initiated within and penetrate from root hairs and deliver the endosymbionts into nodule cells. Despite recent progress in understanding the mutual recognition and early symbiotic signaling cascades in host legumes, the molecular mechanisms underlying bacterial infection processes and successive nodule organogenesis are still poorly understood. We isolated a novel symbiotic mutant of Lotus japonicus, cerberus, which shows defects in IT formation and nodule organogenesis. Map-based cloning of the causal gene allowed us to identify the CERBERUS gene, which encodes a novel protein containing a U-box domain and WD-40 repeats. CERBERUS expression was detected in the roots and nodules, and was enhanced after inoculation of Mesorhizobium loti. Strong expression was detected in developing nodule primordia and the infected zone of mature nodules. In cerberus mutants, Rhizobium colonized curled root hair tips, but hardly penetrated into root hair cells. The occasional ITs that were formed inside the root hair cells were mostly arrested within the epidermal cell layer. Nodule organogenesis was aborted prematurely, resulting in the formation of a large number of small bumps which contained no endosymbiotic bacteria. These phenotypic and genetic analyses, together with comparisons with other legume mutants with defects in IT formation, indicate that CERBERUS plays a critical role in the very early steps of IT formation as well as in growth and differentiation of nodules.