Many insects that rely on a single food source throughout their developmental cycle harbor beneficial microbes that provide nutrients absent from their restricted diet. Tsetse flies, the vectors of African trypanosomes, feed exclusively on blood and rely on one such intracellular microbe for nutritional provisioning and fecundity. As a result of co-evolution with hosts over millions of years, these mutualists have lost the ability to survive outside the sheltered environment of their host insect cells. We present the complete annotated genome of Wigglesworthia glossinidia brevipalpis, which is composed of one chromosome of 697,724 base pairs (bp) and one small plasmid, called pWig1, of 5,200 bp. Genes involved in the biosynthesis of vitamin metabolites, apparently essential for host nutrition and fecundity, have been retained. Unexpectedly, this obligate's genome bears hallmarks of both parasitic and free-living microbes, and the gene encoding the important regulatory protein DnaA is absent.Many arthropods with restricted diets, such as vertebrate blood, plant juice or wood, rely on symbiotic microorganisms to supply nutrients required for viability and fertility 1 . Among insects harboring such symbionts is the tsetse fly (Diptera: Glossinidae)-the vector of African trypanosomes, agents of deadly diseases in humans and animals in sub-Saharan Africa 2 . Tsetse flies harbor two symbiotic microorganisms in gut tissue: the obligate primary-symbiont Wigglesworthia glossinidia and the commensal secondarysymbiont Sodalis glossinidius. Whereas S. glossinidius may be found in various host tissue types, W. glossinidia is housed in differentiated host epithelial cells (bacteriocytes) that form the bacteriome organ 2 . The functional role of obligate symbionts in tsetses has been difficult to study, as their elimination results in retarded growth and a decrease in egg production and fecundity in the aposymbiotic host 3,4 . The ability to reproduce could be partially restored, however, when aposymbiotic flies received supplementation with B-complex vitamins, suggesting that the endosymbionts might have a metabolic role involving these compounds 5 .The phylogenetic characterization of W. glossinidia from distant tsetse species has shown that they form a distinct clade in the Enterobacteriaceae 6 and display concordant evolution with their host species 7 . This finding implies that a tsetse ancestor was infected with a bacterium some 50-100 million years ago, and extant species of tsetse and associated W. glossinidia strains radiated without horizontal transfer of genetic material between species.As a result of their intracellular lifestyle, the genomes of obligate symbionts have undergone massive reductions in comparison with their free-living relatives. The genome size of W. glossinidia has been estimated as 740-770 kilobases 8 (kb), and that of Buchnera sp., the obligate symbiont of the pea aphid (Homoptera:Aphidoidea), as 640,681 bp 9,10 . Both genomes approach the size of the smallest genome reported thus far, that of My...
Pilus biogenesis on the surface of uropathogenic Escherichia coli requires the chaperone/usher pathway, a terminal branch of the general secretory pathway. In this pathway, periplasmic chaperone-subunit complexes target an outer membrane (OM) usher for subunit assembly into pili and secretion to the cell surface. The molecular mechanisms of protein secretion across the OM are not well understood. Mutagenesis of the P pilus usher PapC and the type 1 pilus usher FimD was undertaken to elucidate the initial stages of pilus biogenesis at the OM. Deletion of residues 2 to 11 of the mature PapC N terminus abolished the targeting of the usher by chaperone-subunit complexes and rendered PapC nonfunctional for pilus biogenesis. Similarly, an intact FimD N terminus was required for chaperone-subunit binding and pilus biogenesis. Analysis of PapC-FimD chimeras and N-terminal fragments of PapC localized the chaperone-subunit targeting domain to the first 124 residues of PapC. Single alanine substitution mutations were made in this domain that blocked pilus biogenesis but did not affect targeting of chaperone-subunit complexes. Thus, the usher N terminus does not function simply as a static binding site for chaperone-subunit complexes but also participates in subsequent pilus assembly events.
A sequence database was created for the Leishmania N-acetylglucosamine-1-phosphate transferase (nagt) gene from 193 independent isolates. PCR products of this single-copy gene were analyzed for restriction fragment length polymorphism based on seven nagt sequences initially available. We subsequently sequenced 77 samples and found 19 new variants (genotypes). Alignment of all 26 nagt sequences is gap free, except for a single codon addition or deletion. Phylogenetic analyses of the sequences allow grouping the isolates into three subgenera, each consisting of recognized species complexes, i.e., subgenus Leishmania (L. amazonensis-L. mexicana, L. donovani-L. infantum, L. tropica, L. major, and L. turanica-L. gerbilli), subgenus Viannia (L. braziliensis, L. panamensis), and one unclassified (L. enriettii) species. This hierarchy of grouping is also supported by sequence analyses of selected samples for additional single-copy genes present on different chromosomes. Intraspecies divergence of nagt varies considerably with different species complexes. Interestingly, species complexes with less subspecies divergence are more widely distributed than those that are more divergent. The relevance of this to Leishmania evolutionary adaptation is discussed. Heterozygosity of subspecies variants contributes to intraspecies diversity, which is prominent in L. tropica but not in L. donovani-L. infantum. This disparity is thought to result from the genetic recombination of the respective species at different times as a rare event during their predominantly clonal evolution. Phylogenetically useful sites of nagt are restricted largely to several extended hydrophilic loops predicted from hypothetical models of Leishmania NAGT as an endoplasmic reticulum transmembrane protein. In silico analyses of nagt from fungi and other protozoa further illustrate the potential value of this and, perhaps, other similar transmembrane molecules for phylogenetic analyses of single-cell eukaryotes.Many microorganisms speciate via clonal evolution. They replicate asexually, with genetic recombination as a rare event.A typical example among single-cell eukaryotes is the trypanosomatid protozoa (58, 59), which are mostly parasites, e.g., Leishmania spp. and Trypanosoma spp. Leishmania spp. live extracellularly in the digestive tracts of blood-sucking female sand flies of various species as their vectors and intracellularly in the macrophages of different mammalian hosts, i.e., human, canine, rodent, and other reservoir animals. The complexities of such unusual ecological niches undoubtedly contribute to Leishmania speciation.A large body of biological, biochemical, immunological, and molecular data (7,10,23,55) exists in the literature suggesting that the genus Leishmania consists of three groups (55) as follows: (i) subgenus Leishmania, which includes species complexes distributed in both the New World and the Old World, e.g., L. majorturanica-L. gerbilli; (ii) subgenus Viannia, whose members are restricted to the Neotropics, e.g., L. braziliensis [...
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