The origin and evolution of the thousands of species-specific genes with unknown functions, the so-called orphan genes, has been a mystery. Here, we have studied the rates and patterns of orphan sequence evolution, using the Rickettsia as our reference system. Of the Rickettsia conorii orphans examined in this study, 80% were found to be short gene fragments or fusions of short segments from neighboring genes. We reconstructed the putative sequences of the full-length genes from which the short orphan fragments are thought to have originated. One of the genes thus reconstructed displays weak similarity to the ankyrin-repeat protein family, an identification that is strongly supported by comparative molecular modeling. Studies of the patterns of gene fragmentation underscore the importance of short repeated sequences as targets for recombination events that result in sequence loss and the formation of short, transient open reading frames. Our analysis demonstrates that gene sequences present in the common ancestor can be inferred even in cases when no full-length open reading frame is present in any of the contemporary species. Such reconstructions support the identification of lost protein functions and hint at important lifestyle changes.

Membrane proteins that transport ATP and ADP have been identified in mitochondria, plastids, and obligate intracellular parasites. The mitochondrial ATP/ADP transporters are derived from a broad-specificity transport family of eukaryotic origin, whereas the origin of the plastid/parasite ATP/ADP translocase is more elusive. Here we present the sequences of five genes coding for ATP/ADP translocases from four species of Rickettsia. The results are consistent with an early duplication and divergence of the five ATP/ADP translocases within the rickettsial lineage. A comparison of the phylogenetic depths of the mitochondrial and the plastid/parasite ATP/ADP translocases indicates a deep origin for both transporters. The results provide no evidence for a recent acquisition of the ATP/ADP transporters in Rickettsia via horizontal gene transfer, as previously suggested. A possible function of the two types of ATP/ADP translocases was to allow switches between glycolysis and aerobic respiration in the early eukaryotic cell and its endosymbiont.

An exceptional disposition of the elongation factor genes is observed in Rickettsia prowazekii, in which there is only one tuf gene, which is distant from the lone fus gene. In contrast, the closely related bacterium Agrobacterium tumefaciens has the normal bacterial arrangement of two tuf genes, of which one is tightly linked to the fus gene. Analysis of the flanking sequences of the single tuf gene in R. prowazekii shows that it is preceded by two of the four tRNA genes located in the 5 region of the Escherichia coli tufB gene and that it is followed by rpsJ as well as associated ribosomal protein genes, which in E. coli are located downstream of the tufA gene. The fus gene is located within the str operon and is followed by one tRNA gene as well as by the genes secE and nusG, which are located in the 3 region of tufB in E. coli. This atypical disposition of genes suggests that intrachromosomal recombination between duplicated tuf genes has contributed to the evolution of the unique genomic architecture of R. prowazekii.Bacteria that are obligate parasites of eucaryotic cells, such as Mycoplasma, Coxiella, Chlamydia, and Rickettsia spp., are often relatively small, with genomes of the size of 1 Mb or less. The phylogenetic placement of these bacteria on the basis of their rRNA genes indicate that they are independent descendents of diverse bacteria with much larger genomes (40). In order to examine the hypothesis that reductive evolution of the genomes of obligate parasitic bacteria proceeds through intrachromosomal recombination at duplicated genes, we have compared the dispositions of the genes coding for the elongation factors in the alpha proteobacteria Rickettsia prowazekii and Agrobacterium tumefaciens.These closely related bacteria differ drastically in their lifestyles and genomic architectures. R. prowazekii is an obligate intracellular parasite causing typhus in humans, and it has a single, circular genome of 1.1 Mb (16). In contrast, A. tumefaciens is a free-living soil bacterium that is also responsible for the development of crown gall and hairy root diseases of dicotyledonous plants (13,63). It has a large, complex genome containing four replicons: two chromosomes of 3.0 and 2.1 Mb, a 450-kb cryptic plasmid, and the 200-kb Ti plasmid (1).The gene for elongation factor Tu (EF-Tu) is present in two copies in the genome of Escherichia coli (18, 23) as well as in many other proteobacterial genomes (54). Furthermore, the two tuf genes are normally associated with characteristic flanking sequences. For example, in E. coli, tufA is a member of the str (streptomycin) operon, in which the genes are arranged in the order rpsL, rpsG, fusA, and tufA, whereas tufB is part of the tufB operon, in which four tRNA genes, thrU, tyrU, glyT, and thrT, are positioned at the 5Ј side of the tufB gene (3,46,62). Similar arrangements of the flanking sequences of tuf genes are common in eubacteria and in archaebacteria (6,25,42). Such widely preserved patterns of genomic disposition presumably reflect ancient ancestral pa...

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A global survey of microbial genomes reveals a correlation between genome size, repeat content and lifestyle. Free-living bacteria have large genomes with a high content of repeated sequences and self-propagating DNA, such as transposons and bacteriophages. In contrast, obligate intracellular bacteria have small genomes with a low content of repeated sequences and no or few genetic parasites. In extreme cases, such as in the 650 kb-genomes of aphid endosymbionts of the genus Buchnera all repeated sequences above 200bp have been eliminated. We speculate that the initial downsizing of the genomes of obligate symbionts and parasites occurred by homologous recombination at repeated genes, leading to the loss of large blocks of DNA as well as to the consumption of repeated sequences. Further sequence elimination in these small genomes seems primarily to result from the accumulation of short deletions within genic sequences. This process may lead to temporary increases in the genomic content of pseudogenes and 'junk' DNA. We discuss causes and long-term consequences of extreme genome size reductions in obligate intracellular bacteria.