The Widespread Colonization Island of Actinobacillus actinomycetemcomitans

Genomic islands, such as pathogenicity islands, contribute to the evolution and diversification of microbial life. Here we report on the Widespread Colonization Island, which encompasses the tad (tight adherence) locus for colonization of surfaces and biofilm formation by the human pathogen Actinobacillus actinomycetemcomitans. At least 12 of the 14 genes at the tad locus are required for tenacious biofilm formation and synthesis of bundled Flp pili (fibrils) that mediate adherence. The pilin subunit, Flp1, remains inside the cell in tad-locus mutants, indicating that these genes encode a secretion system for export and assembly of fibrils. We found tad-related regions in a wide variety of Bacterial and Archaeal species, and their sequence characteristics indicate possible horizontal transfer. To test the hypothesis of horizontal transfer, we compared the phylogeny of the tad locus to a robust organismal phylogeny using statistical tests of congruence and tree reconciliation techniques. Our analysis strongly supports a complex history of gene shuffling by recombination and multiple horizontal transfers, duplications and losses. We present evidence for a specific horizontal transfer event leading to the establishment of this region as a determinant of disease.     

MAGIC, an in vivo genetic method for the rapid construction of recombinant DNA molecules

We describe a highly engineered in vivo cloning method, mating-assisted genetically integrated cloning (MAGIC), that facilitates the rapid construction of recombinant DNA molecules. MAGIC uses bacterial mating, in vivo site-specific endonuclease cleavage and homologous recombination to catalyze the transfer of a DNA fragment between a donor vector in one bacterial strain and a recipient plasmid in a separate bacterial strain. Recombination events are genetically selected and result in placement of the gene of interest under the control of new regulatory elements with high efficiency. MAGIC eliminates the need for restriction enzymes, DNA ligases, preparation of DNA and all in vitro manipulations required for subcloning and allows the rapid construction of multiple constructs with minimal effort. We show that MAGIC can generate constructs for expression in multiple organisms. As this new method requires only the simple mixing of bacterial strains, it represents a substantial advance in high-throughput recombinant DNA production that will save time, effort and expense in functional genomics studies.     

Complementation cloning identifies CDG-IIc, a new type of congenital disorders of glycosylation, as a GDP-fucose transporter deficiency

Congenital disorders of glycosylation (CDG) comprise a rapidly growing group of inherited disorders in which glycosylation of glycoproteins is defective due to mutations in genes required for the assembly of lipid-linked oligosaccharides, their transfer to nascent glycoproteins (CDG-I) or the processing of protein-bound glycans (CDG-II). Previously' a defect in the GDP-fucose import into the lumen of the Golgi was identified in a person with CDG (A.C.) with a general deficiency of fucosyl residues in glycoproteins. This patient presents the clinical features of leukocyte adhesion deficiency type II (LAD II) including mental retardation, short stature, facial stigmata, and recurrent bacterial peripheral infections with persistently elevated peripheral leukocytes. Using a fucose-specific, lectin-staining procedure for detection of fucosylated glycoproteins and a retroviral cDNA library, we isolated a cDNA complementing the fucosylation defect in the patient's fibroblasts. The cDNA encodes a highly hydrophobic protein of 364 amino acids with multiple putative transmembrane domains. Restoration of GDP-fucose import activity in Golgi-enriched vesicles from the patient's fibroblasts verified the GDP-fucose transporter activity of this protein. We identified two missense mutations in the GDP-fucose transporter cDNA of patient A.C. and of two other people with LAD II. Thus complementation cloning allowed us to identify the human GDP-fucose transporter cDNA and GDP-fucose transporter deficiency as a cause for a new type of CDG. Following the recent recommendations for the nomenclature for CDG, this new type is classified as CDG-IIc (formerly LAD II).     

Emergence of a new disease as a result of interspecific virulence gene transfer

New diseases of humans, animals and plants emerge regularly. Enhanced virulence on a new host can be facilitated by the acquisition of novel virulence factors. Interspecific gene transfer is known to be a source of such virulence factors in bacterial pathogens (often manifested as pathogenicity islands in the recipient organism) and it has been speculated that interspecific transfer of virulence factors may occur in fungal pathogens. Until now, no direct support has been available for this hypothesis. Here we present evidence that a gene encoding a critical virulence factor was transferred from one species of fungal pathogen to another. This gene transfer probably occurred just before 1941, creating a pathogen population with significantly enhanced virulence and leading to the emergence of a new damaging disease of wheat.     

Mutations in the human homologue of mouse dl cause autosomal recessive and dominant hypohidrotic ectodermal dysplasia.

X-linked hypohidrotic ectodermal dysplasia results in abnormal morphogenesis of teeth, hair and eccrine sweat glands. The gene (ED1) responsible for the disorder has been identified, as well as the analogous X-linked gene (Ta) in the mouse. Autosomal recessive disorders, phenotypically indistinguishable from the X-linked forms, exist in humans and at two separate loci (crinkled, cr, and downless, dl) in mice. Dominant disorders, possibly allelic to the recessive loci, are seen in both species (ED3, Dlslk). A candidate gene has recently been identified at the dl locus that is mutated in both dl and Dlslk mutant alleles. We isolated and characterized its human DL homologue, and identified mutations in three families displaying recessive inheritance and two with dominant inheritance. The disorder does not map to the candidate gene locus in all autosomal recessive families, implying the existence of at least one additional human locus. The putative protein is predicted to have a single transmembrane domain, and shows similarity to two separate domains of the tumour necrosis factor receptor (TNFR) family.     

Comparable system-level organization of Archaea and Eukaryotes

A central and long-standing issue in evolutionary theory is the origin of the biological variation upon which natural selection acts. Some hypotheses suggest that evolutionary change represents an adaptation to the surrounding environment within the constraints of an organism's innate characteristics. Elucidation of the origin and evolutionary relationship of species has been complemented by nucleotide sequence and gene content analyses, with profound implications for recognizing life's major domains. Understanding of evolutionary relationships may be further expanded by comparing systemic higher-level organization among species. Here we employ multivariate analyses to evaluate the biochemical reaction pathways characterizing 43 species. Comparison of the information transfer pathways of Archaea and Eukaryotes indicates a close relationship between these domains. In addition, whereas eukaryotic metabolic enzymes are primarily of bacterial origin, the pathway-level organization of archaeal and eukaryotic metabolic networks is more closely related. Our analyses therefore suggest that during the symbiotic evolution of eukaryotes, incorporation of bacterial metabolic enzymes into the proto-archaeal proteome was constrained by the host's pre-existing metabolic architecture.     

Biased biological functions of horizontally transferred genes in prokaryotic genomes

Horizontal gene transfer is one of the main mechanisms contributing to microbial genome diversification. To clarify the overall picture of interspecific gene flow among prokaryotes, we developed a new method for detecting horizontally transferred genes and their possible donors by Bayesian inference with training models for nucleotide composition. Our method gives the average posterior probability (horizontal transfer index) for each gene sequence, with a low horizontal transfer index indicating recent horizontal transfer. We found that 14% of open reading frames in 116 prokaryotic complete genomes were subjected to recent horizontal transfer. Based on this data set, we quantitatively determined that the biological functions of horizontally transferred genes, except mobile element genes, are biased to three categories: cell surface, DNA binding and pathogenicity-related functions. Thus, the transferability of genes seems to depend heavily on their functions.     

Adaptive evolution of bacterial metabolic networks by horizontal gene transfer

Numerous studies have considered the emergence of metabolic pathways, but the modes of recent evolution of metabolic networks are poorly understood. Here, we integrate comparative genomics with flux balance analysis to examine (i) the contribution of different genetic mechanisms to network growth in bacteria, (ii) the selective forces driving network evolution and (iii) the integration of new nodes into the network. Most changes to the metabolic network of Escherichia coli in the past 100 million years are due to horizontal gene transfer, with little contribution from gene duplicates. Networks grow by acquiring genes involved in the transport and catalysis of external nutrients, driven by adaptations to changing environments. Accordingly, horizontally transferred genes are integrated at the periphery of the network, whereas central parts remain evolutionarily stable. Genes encoding physiologically coupled reactions are often transferred together, frequently in operons. Thus, bacterial metabolic networks evolve by direct uptake of peripheral reactions in response to changed environments.     

Merlin--rapid analysis of dense genetic maps using sparse gene flow trees.

Efforts to find disease genes using high-density single-nucleotide polymorphism (SNP) maps will produce data sets that exceed the limitations of current computational tools. Here we describe a new, efficient method for the analysis of dense genetic maps in pedigree data that provides extremely fast solutions to common problems such as allele-sharing analyses and haplotyping. We show that sparse binary trees represent patterns of gene flow in general pedigrees in a parsimonious manner, and derive a family of related algorithms for pedigree traversal. With these trees, exact likelihood calculations can be carried out efficiently for single markers or for multiple linked markers. Using an approximate multipoint calculation that ignores the unlikely possibility of a large number of recombinants further improves speed and provides accurate solutions in dense maps with thousands of markers. Our multipoint engine for rapid likelihood inference (Merlin) is a computer program that uses sparse inheritance trees for pedigree analysis; it performs rapid haplotyping, genotype error detection and affected pair linkage analyses and can handle more markers than other pedigree analysis packages.     

De novo mutations in the gene encoding STXBP1 (MUNC18-1) cause early infantile epileptic encephalopathy

Early infantile epileptic encephalopathy with suppression-burst (EIEE), also known as Ohtahara syndrome, is one of the most severe and earliest forms of epilepsy. Using array-based comparative genomic hybridization, we found a de novo 2.0-Mb microdeletion at 9q33.3-q34.11 in a girl with EIEE. Mutation analysis of candidate genes mapped to the deletion revealed that four unrelated individuals with EIEE had heterozygous missense mutations in the gene encoding syntaxin binding protein 1 (STXBP1). STXBP1 (also known as MUNC18-1) is an evolutionally conserved neuronal Sec1/Munc-18 (SM) protein that is essential in synaptic vesicle release in several species. Circular dichroism melting experiments revealed that a mutant form of the protein was significantly thermolabile compared to wild type. Furthermore, binding of the mutant protein to syntaxin was impaired. These findings suggest that haploinsufficiency of STXBP1 causes EIEE.     

Exome sequencing identifies truncating mutations in PRRT2 that cause paroxysmal kinesigenic dyskinesia

Paroxysmal kinesigenic dyskinesia is the most common type of paroxysmal movement disorder and is often misdiagnosed clinically as epilepsy. Using whole-exome sequencing followed by Sanger sequencing, we identified three truncating mutations within PRRT2 (NM_145239.2) in eight Han Chinese families with histories of paroxysmal kinesigenic dyskinesia: c.514_517delTCTG (p.Ser172Argfs*3) in one family, c.649dupC (p.Arg217Profs*8) in six families and c.972delA (p.Val325Serfs*12) in one family. These truncating mutations co-segregated exactly with the disease in these families and were not observed in 1,000 control subjects of matched ancestry. PRRT2 is a newly discovered gene consisting of four exons encoding the proline-rich transmembrane protein 2, which encompasses 340 amino acids and contains two predicted transmembrane domains. PRRT2 is highly expressed in the developing nervous system, and a truncating mutation alters the subcellular localization of the PRRT2 protein. The function of PRRT2 and its role in paroxysmal kinesigenic dyskinesia should be further investigated.     

Modularity and evolutionary constraint on proteins

Modularity, which has been found in the functional and physical protein interaction networks of many organisms, has been postulated to affect both the mode and tempo of evolution. Here I show that in the yeast Saccharomyces cerevisiae, protein interaction hubs situated in single modules are highly constrained, whereas those connecting different modules are more plastic. This pattern of change could reflect a tendency for evolutionary innovations to occur by altering the proteins and interactions between rather than within modules, in a manner somewhat similar to the evolution of new proteins through the shuffling of conserved protein domains.     

Duplication-degeneration as a mechanism of gene fission and the origin of new genes in Drosophila species

Gene fission and fusion, the processes by which a single gene is split into two separate genes and two adjacent genes are fused into a single gene, respectively, are among the primary processes that generate new genes. Despite their seeming reversibility, nothing is known about the mechanism of gene fission. Because the nucleotide sequences of fission genes record little about their origination process, conventional analysis of duplicate genes may not be powerful enough to unravel the underlying mechanism. In a survey for young genes in species of the Drosophila melanogaster subgroup using fluorescence in situ hybridization, we identified a young gene family, monkey king, whose genesis sheds light on the evolutionary process of gene fission. Its members originated 1-2 million years ago as retroposed duplicates and evolved into fission genes that separately encode protein domains from a multidomain ancestor. The mechanism underlying this process is gene duplication with subsequent partial degeneration.