The archaeal flagellum: a different kind of prokaryotic motility structure

The archaeal flagellum is a unique motility apparatus distinct in composition and likely in assembly from the bacterial flagellum. Gene families comprised of multiple flagellin genes co-transcribed with a number of conserved, archaeal-specific accessory genes have been identified in several archaea. However, no homologues of any bacterial genes involved in flagella structure have yet been identified in any archaeon, including those archaea in which the complete genome sequence has been published. Archaeal flagellins possess a highly conserved hydrophobic N-terminal sequence that is similar to that of type IV pilins and clearly unlike that of bacterial flagellins. Also unlike bacterial flagellins but similar to type IV pilins, archaeal flagellins are initially synthesized with a short leader peptide that is cleaved by a membrane-located peptidase. With recent advances in genetic transfer systems in archaea, knockouts have been reported in several genes involved in flagellation in different archaea. In addition, techniques to isolate flagella with attached hook and anchoring structures have been developed. Analysis of these preparations is under way to identify minor structural components of archaeal flagella. This and the continued isolation and characterization of flagella mutants should lead to significant advances in our knowledge of the composition and assembly of archaeal flagella.     

Recent advances in the structure and assembly of the archaeal flagellum

Archaeal motility occurs through the rotation of flagella that are distinct from the flagella found on bacteria. The differences between the two structures include the multi-flagellin nature of the archaeal filament, the widespread posttranslational modification of the flagellins and the presence of a short signal peptide on each flagellin that is cleaved by a specific signal peptidase prior to the incorporation of the mature flagellin into the flagellar filament. Research has revealed similarities between the archaeal flagellum and the type IV pilus, including the presence of similar unusual signal peptides on the flagellins and pilins, similarities in the amino acid sequences of the major structural proteins themselves, as well as similarities between potential assembly and processing components. The recent suggestion that type IV pili are part of a family of cell surface complexes, coupled with the similarities between type IV pili and archaeal flagella, raise questions about the evolution of these systems and possible inclusion of archaeal flagella into this surface complex family.     

Assembly and function of the archaeal flagellum

Motility is a common behaviour in prokaryotes. Both bacteria and archaea use flagella for swimming motility, but it has been well documented that structures of the flagellum from these two domains of life are completely different, although they contribute to a similar function. Interestingly, information available to date has revealed that structurally archaeal flagella are more similar to bacterial type?IV pili rather than to bacterial flagella. With the increasing genome sequence information and advancement in genetic tools for archaea, identification of the components involved in the assembly of the archaeal flagellum is possible. A subset of these components shows similarities to components from type?IV pilus-assembly systems. Whereas the molecular players involved in assembly of the archaeal flagellum are being identified, the mechanics and dynamics of the assembly of the archaeal flagellum have yet to be established. Recent computational analysis in our laboratory has identified conserved highly charged loop regions within one of the core proteins of the flagellum, the membrane integral protein FlaJ, and predicted that these are involved in the interaction with the assembly ATPase FlaI. Interestingly, considerable variation was found among the loops of FlaJ from the two major subkingdoms of archaea, the Euryarchaeota and the Crenarchaeota. Understanding the assembly pathway and creating an interaction map of the molecular players in the archaeal flagellum will shed light on the details of the assembly and also the evolutionary relationship to the bacterial type?IV pili-assembly systems.     

Tuberculina-rusts: a unique basidiomycetous interfungal cellular interaction with horizontal nuclear transfer

Cellular interaction of the basidiomycete Tuberculina persicina with the haploid stages of two rusts Puccinia silvatica and Tranzschelia pruni-spinosae was analyzed by serial-section electron microscopy of chemically fixed samples and samples subjected to high-pressure freezing and freeze substitution. Tuberculina persicina is a contact parasite, forming neither haustoria nor other intracellular structures. However, at contact areas between T. persicina and its hosts, distinct interfungal interactions are present. At the beginning, a hyphal cell of T. persicina invades the host cell wall with a protuberance and the cell walls of both protuberance and host cell dissolve at the point of contact. Thus, the plasma membranes of the two organisms contact and fuse to form a pore that enlarges to a final diameter of approximately 1 μm. The membrane of the fusion pore is continuous with the plasma membranes of both cells, and Tuberculina nuclei and other organelles are transferred to the rust cells. Phylogenetic and functional aspects of this curious basidiomycetous interfungal interaction are discussed, and a hypothesis of the evolutionary derivation of the Tuberculina mycoparasitism from a sexual interaction is presented.     

Crystal structure of the flagellar accessory protein FlaH of Methanocaldococcus jannaschii suggests a regulatory role in archaeal flagellum assembly

Archaeal flagella are unique structures that share functional similarity with bacterial flagella, but are structurally related to bacterial type IV pili. The flagellar accessory protein FlaH is one of the conserved components of the archaeal motility system. However, its function is not clearly understood. Here, we present the 2.2 Å resolution crystal structure of FlaH from the hyperthermophilic archaeon, Methanocaldococcus jannaschii. The protein has a characteristic RecA‐like fold, which has been found previously both in archaea and bacteria. We show that FlaH binds to immobilized ATP—however, it lacks ATPase activity. Surface plasmon resonance analysis demonstrates that ATP affects the interaction between FlaH and the archaeal motor protein FlaI. In the presence of ATP, the FlaH‐FlaI interaction becomes significantly weaker. A database search revealed similarity between FlaH and several DNA‐binding proteins of the RecA superfamily. The closest structural homologs of FlaH are KaiC‐like proteins, which are archaeal homologs of the circadian clock protein KaiC from cyanobacteria. We propose that one of the functions of FlaH may be the regulation of archaeal motor complex assembly.     

Targeted disruption of the Akap4 gene causes defects in sperm flagellum and motility

A-kinase anchoring proteins (AKAPs) tether cyclic AMP-dependent protein kinases and thereby localize phosphorylation of target proteins and initiation of signal-transduction processes triggered by cyclic AMP. AKAPs can also be scaffolds for kinases and phosphatases and form macromolecular complexes with other proteins involved in signal transduction. Akap4 is transcribed only in the postmeiotic phase of spermatogenesis and encodes the most abundant protein in the fibrous sheath, a novel cytoskeletal structure present in the principal piece of the sperm flagellum. Previous studies indicated that cyclic AMP-dependent signaling processes are important in the regulation of sperm motility, and gene targeting was used here to test the hypothesis that AKAP4 is a scaffold for protein complexes involved in regulating flagellar function. Sperm numbers were not reduced in male mice lacking AKAP4, but sperm failed to show progressive motility and male mice were infertile. The fibrous sheath anlagen formed, but the definitive fibrous sheath did not develop, the flagellum was shortened, and proteins usually associated with the fibrous sheath were absent or substantially reduced in amount. However, the other cytoskeletal components of the flagellum were present and appeared fully developed. We conclude that AKAP4 is a scaffold protein required for the organization and integrity of the fibrous sheath and that effective sperm motility is lost in the absence of AKAP4 because signal transduction and glycolytic enzymes fail to become associated with the fibrous sheath.     

Distant horizontal gene transfer is rare for multiple families of prokaryotic insertion sequences

Horizontal gene transfer in prokaryotes is rampant on short and intermediate evolutionary time scales. It poses a fundamental problem to our ability to reconstruct the evolutionary tree of life. Is it also frequent over long evolutionary distances? To address this question, we analyzed the evolution of 2,091 insertion sequences from all 20 major families in 438 completely sequenced prokaryotic genomes. Specifically, we mapped insertion sequence occurrence on a 16S rDNA tree of the genomes we analyzed, and we also constructed phylogenetic trees of the insertion sequence transposase coding sequences. We found only 30 cases of likely horizontal transfer among distantly related prokaryotic clades. Most of these horizontal transfer events are ancient. Only seven events are recent. Almost all of these transfer events occur between pairs of human pathogens or commensals. If true also for other, non-mobile DNA, the rarity of distant horizontal transfer increases the odds of reliable phylogenetic inference from sequence data.     

Towards a quantitative understanding of horizontal gene transfer: a kinetic model

In this work, we present a simple kinetic model of horizontal gene transfer. It describes the processes of gene duplication, mutation, gene transfer and the regulation of the total size of the genome for genetically homogeneous prokaryotic species or strains. The emerging nonlinear system of first-order differential equations can be linearized at the stationary point. For selected models, we give an analytical solution for the number of foreign and native genes within a species. We identify a regime characterized by a fast gene transfer rate and species with a mixed genome, a slow gene transfer regime with pure organisms, and a crossover region. The data are compared to experiments, and the biological implications of our model are discussed.     

DarkHorse: a method for genome-wide prediction of horizontal gene transfer

A new approach to rapid, genome-wide identification and ranking of horizontal transfer candidate proteins is presented. The method is quantitative, reproducible, and computationally undemanding. It can be combined with genomic signature and/or phylogenetic tree-building procedures to improve accuracy and efficiency. The method is also useful for retrospective assessments of horizontal transfer prediction reliability, recognizing orthologous sequences that may have been previously overlooked or unavailable. These features are demonstrated in bacterial, archaeal, and eukaryotic examples.     

Phylogenomics of T4 cyanophages: lateral gene transfer in the 'core' and origins of host genes

The last two decades have revealed that phages (viruses that infect bacteria) are abundant and play fundamental roles in the Earth System, with the T4-like myoviruses (herein T4-like phages) emerging as a dominant 'signal' in wild populations. Here we examine 27 T4-like phage genomes, with a focus on 17 that infect ocean picocyanobacteria (cyanophages), to evaluate lateral gene transfer (LGT) in this group. First, we establish a reference tree by evaluating concatenated core gene supertrees and whole genome gene content trees. Next, we evaluate what fraction of these 'core genes' shared by all 17 cyanophages appear prone to LGT. Most (47 out of 57 core genes) were vertically transferred as inferred from tree tests and genomic synteny. Of those 10 core genes that failed the tree tests, the bulk (8 of 10) remain syntenic in the genomes with only a few (3 of the 10) having identifiable signatures of mobile elements. Notably, only one of these 10 is shared not only by the 17 cyanophages, but also by all 27 T4-like phages (thymidylate synthase); its evolutionary history suggests cyanophages may be the origin of these genes to Prochlorococcus. Next, we examined intragenic recombination among the core genes and found that it did occur, even among these core genes, but that the rate was significantly higher between closely related phages, perhaps reducing any detectable LGT signal and leading to taxon cohesion. Finally, among 18 auxiliary metabolic genes (AMGs, a.k.a. 'host' genes), we found that half originated from their immediate hosts, in some cases multiple times (e.g. psbA, psbD, pstS), while the remaining have less clear evolutionary origins ranging from cyanobacteria (4 genes) or microbes (5 genes), with particular diversity among viral TalC and Hsp20 sequences. Together, these findings highlight the patterns and limits of vertical evolution, as well as the ecological and evolutionary roles of LGT in shaping T4-like phage genomes.     

Plasmid-mediated horizontal gene transfer is a coevolutionary process

Conjugative plasmids are key agents of horizontal gene transfer (HGT) that accelerate bacterial adaptation by vectoring ecologically important traits between strains and species. However, although many conjugative plasmids carry beneficial traits, all plasmids exert physiological costs-of-carriage on bacteria. The existence of conjugative plasmids, therefore, presents a paradox because non-beneficial plasmids should be lost to purifying selection, whereas beneficial genes carried on plasmids should be integrated into the bacterial chromosome. Several ecological solutions to the paradox have been proposed, but none account for co-adaptation of bacteria and conjugative plasmids. Drawing upon evidence from experimental evolution, we argue that HGT via conjugation can only be fully understood in a coevolutionary framework.     

Identification and structure of the Rhizobium galegae common nodulation genes: evidence for horizontal gene transfer

Rhizobia are soil bacteria able to fix atmospheric nitrogen in symbiosis with leguminous plants. In response to a signal cascade coded by genes of both symbiotic partners, a specific plant organ, the nodule, is formed. Rhizobial nodulation (nod) genes trigger nodule formation through the synthesis of Nod factors, a family of chitolipooligosaccharides that are specifically recognized by the host plant at the first stages of the nodulation process. Here, we present the organization and sequence of the common nod genes from Rhizobium galegae, a symbiotic member of the RHIZOBIACEAE: This species has an intriguing phylogenetic position, being symbiotic among pathogenic agrobacteria, which induce tumors instead of nodules in plant shoots or roots. This apparent incongruence raises special interest in the origin of the symbiotic apparatus of R. galegae. Our analysis of DNA sequence data indicated that the organization of the common nod gene region of R. galegae was similar to that of Sinorhizobium meliloti and Rhizobium leguminosarum, with nodIJ downstream of nodABC and the regulatory nodD gene closely linked to the common nod operon. Moreover, phylogenetic analyses of the nod gene sequences showed a close relationship especially between the common nodA sequences of R. galegae, S. meliloti, and R. leguminosarum biovars viciae and trifolii. This relationship in structure and sequence contrasts with the phylogeny based on 16S rRNA, which groups R. galegae close to agrobacteria and separate from most other rhizobia. The topology of the nodA tree was similar to that of the corresponding host plant tree. Taken together, these observations indicate that lateral nod gene transfer occurred from fast-growing rhizobia toward agrobacteria, after which the symbiotic apparatus evolved under host plant constraint.     

Phylogeny vs genome reshuffling: horizontal gene transfer

The evolutionary events in organisms can be tracked to the transfer of genetic material. The inheritance of genetic material among closely related organisms is a slow evolutionary process. On the other hand, the movement of genes among distantly related species can account for rapid evolution. The later process has been quite evident in the appearance of antibiotic resistance genes among human and animal pathogens. Phylogenetic trees based on such genes and those involved in metabolic activities reflect the incongruencies in comparison to the 16S rDNA gene, generally used for taxonomic relationships. Such discrepancies in gene inheritance have been termed as horizontal gene transfer (HGT) events. In the post-genomic era, the explosion of known sequences through large-scale sequencing projects has unraveled the weakness of traditional 16S rDNA gene tree based evolutionary model. Various methods to scrutinize HGT events include atypical composition, abnormal sequence similarity, anomalous phylogenetic distribution, unusual phyletic patterns, etc. Since HGT generates greater genetic diversity, it is likely to increase resource use and ecosystem resilience.     

Detecting highways of horizontal gene transfer

In a horizontal gene transfer (HGT) event, a gene is transferred between two species that do not have an ancestor-descendant relationship. Typically, no more than a few genes are horizontally transferred between any two species. However, several studies identified pairs of species between which many different genes were horizontally transferred. Such a pair is said to be linked by a highway of gene sharing. We present a method for inferring such highways. Our method is based on the fact that the evolutionary histories of horizontally transferred genes disagree with the corresponding species phylogeny. Specifically, given a set of gene trees and a trusted rooted species tree, each gene tree is first decomposed into its constituent quartet trees and the quartets that are inconsistent with the species tree are identified. Our method finds a pair of species such that a highway between them explains the largest (normalized) fraction of inconsistent quartets. For a problem on n species and m input quartet trees, we give an efficient O(m + n(2))-time algorithm for detecting highways, which is optimal with respect to the quartets input size. An application of our method to a dataset of 1128 genes from 11 cyanobacterial species, as well as to simulated datasets, illustrates the efficacy of our method.     

The human serum albumin gene: structure of a unique locus

The entire gene for human serum albumin (HSA) has been isolated from a genomic DNA library, carried in the lambda Charon 4A vector. Six independent isolates have been found to hybridize to a cloned HSA cDNA probe, and all six clones share restriction site sequence homology in the overlapping portion of their DNA. These results seem to indicate that the albumin gene is single-copy, or unique, within the human haploid genome. Measuring from the "CAP" site to the "poly(A)" addition site, albumin gene comprises 16.5 kb of DNA.     

Phylogenetic analysis of the isopenicillin-N-synthetase horizontal gene transfer

A phylogenetic study of the isopenicillin-N-synthetase (IPNS) gene sequence from prokaryotic and lower eukaryotic producers of β-lactam antibiotics by means of a maximum-likelihood approach has been carried out. After performing an extensive search, rather than invoking a global molecular clock, the results obtained are best explained by a model with three rates of evolution. Grouped in decreasing order, these correspond toA. nidulans and then to the rest of the eukaryotes and prokaryotes, respectively. The estimated branching date between prokaryotic and fungal IPNS sequences (852 ±106 MY) strongly supports the hypothesis that the IPNS gene was horizontally transferred from bacterial β-lactam producers to filamentous fungi. Correspondence to: A. Moya     

Insight in to the phylogeny of polyhydroxyalkanoate biosynthesis: horizontal gene transfer

Polyhydroxyalkanoates (PHAs) are gaining more and more importance the world over due to their structural diversity and close analogy to plastics. Their biodegradability makes them extremely desirable substitutes for synthetic plastics. PHAs are produced in organisms under certain stress conditions. Here, we investigated 253 sequenced (completely and unfinished) genomes for the diversity and phylogenetics of the PHA biosynthesis. Discrepancies in the phylogenetic trees for phaA, phaB and phaC genes of the PHA biosynthesis have led to the suggestion that horizontal gene transfer (HGT) may be a major contributor for its evolution. Twenty four organisms belonging to diverse taxa were found to be involved in HGT. Among these, Bacillus cereus ATCC 14579 and Xanthomonas axonopodis pv. citri str. 306 seem to have acquired all the three genes through HGT events and have not been characterized so far as PHA producers. This study also revealed certain potential organisms such as Streptomyces coelicolor A3(2), Staphylococcus epidermidis ATCC 12228, Brucella suis 1330, Burkholderia sp., DSMZ 9242 and Leptospira interrogans serovar lai str. 56601, which can be transformed into novel PHA producers through recombinant DNA technology.