Origin and evolution of the mitochondrial proteome.

The endosymbiotic theory for the origin of mitochondria requires substantial modification. The three identifiable ancestral sources to the proteome of mitochondria are proteins descended from the ancestral alpha-proteobacteria symbiont, proteins with no homology to bacterial orthologs, and diverse proteins with bacterial affinities not derived from alpha-proteobacteria. Random mutations in the form of deletions large and small seem to have eliminated nonessential genes from the endosymbiont-mitochondrial genome lineages. This process, together with the transfer of genes from the endosymbiont-mitochondrial genome to nuclei, has led to a marked reduction in the size of mitochondrial genomes. All proteins of bacterial descent that are encoded by nuclear genes were probably transferred by the same mechanism, involving the disintegration of mitochondria or bacteria by the intracellular membranous vacuoles of cells to release nucleic acid fragments that transform the nuclear genome. This ongoing process has intermittently introduced bacterial genes to nuclear genomes. The genomes of the last common ancestor of all organisms, in particular of mitochondria, encoded cytochrome oxidase homologues. There are no phylogenetic indications either in the mitochondrial proteome or in the nuclear genomes that the initial or subsequent function of the ancestor to the mitochondria was anaerobic. In contrast, there are indications that relatively advanced eukaryotes adapted to anaerobiosis by dismantling their mitochondria and refitting them as hydrogenosomes. Accordingly, a continuous history of aerobic respiration seems to have been the fate of most mitochondrial lineages. The initial phases of this history may have involved aerobic respiration by the symbiont functioning as a scavenger of toxic oxygen. The transition to mitochondria capable of active ATP export to the host cell seems to have required recruitment of eukaryotic ATP transport proteins from the nucleus. The identity of the ancestral host of the alpha-proteobacterial endosymbiont is unclear, but there is no indication that it was an autotroph. There are no indications of a specific alpha-proteobacterial origin to genes for glycolysis. In the absence of data to the contrary, it is assumed that the ancestral host cell was a heterotroph.

Insights into the evolutionary process of genome degradation.

Studies of noncoding and pseudogene sequence diversity, particularly in Rickettsia, have begun to reveal the basic principles of genome degradation in microorganisms. Increasingly, studies of genes and genomes suggest that there has been an extensive amount of horizontal gene transfer among microorganisms. As this inflow of genetic material does not seem generally to have resulted in genome size expansions, however, degenerative processes must be at the very least as widespread as horizontal gene transfer. The basic principles of gene degradation and elimination that are being explored in Rickettsia are likely to be of major importance for our understanding of how microbial genomes evolve.

Origins of mitochondria and hydrogenosomes.

Complete genome sequences for many mitochondria, as well as for some bacteria, together with the nuclear genome sequence of yeast have provided a coherent view of the origin of mitochondria. In particular, conventional phylogenetic reconstructions with genes coding for proteins active in energy metabolism and translation have confirmed the simplest version of the endosymbiosis hypothesis. In contrast, the hydrogen and the syntrophy hypotheses for the origin of mitochondria do not receive support from the available data. It remains to be seen how the evolution of hydrogenosomes is related to that of mitochondria.

A phylogenomic approach to microbial evolution.

To study the origin and evolution of biochemical pathways in microorganisms, we have developed methods and software for automatic, large-scale reconstructions of phylogenetic relationships. We define the complete set of phylogenetic trees derived from the proteome of an organism as the phylome and introduce the term phylogenetic connection as a concept that describes the relative relationships between taxa in a tree. A query system has been incorporated into the system so as to allow searches for defined categories of trees within the phylome. As a complement, we have developed the pyphy system for visualising the results of complex queries on phylogenetic connections, genomic locations and functional assignments in a graphical format. Our phylogenomics approach, which links phylogenetic information to the flow of biochemical pathways within and among microbial species, has been used to examine more than 8000 phylogenetic trees from seven microbial genomes. The results have revealed a rich web of phylogenetic connections. However, the separation of Bacteria and Archaea into two separate domains remains robust.

Reductive evolution of resident genomes.

Small, asexual populations are expected to accumulate deleterious substitutions and deletions in an irreversible manner, which in the long-term will lead to mutational meltdown and genome decay. Here, we discuss the influence of such reductive processes on the evolution of genomes that replicate within the domain of a host genome.

Bioenergetics of the obligate intracellular parasite Rickettsia prowazekii.

Mitochondria are thought to be derived from an ancestor of the alpha-proteobacteria and more specifically from the Rickettsiaceae. The bioenergetic repertoire of the obligate intracellular parasite Rickettsia prowazekii is consistent with its postulated role as the ancestor of the mitochondria. For example, the R. prowazekii genome contains genes encoding components of the tricarboxylic acid cycle as well as of the electron transport system, but lacks genes to support glycolysis. In addition, the R. prowazekii genome contains multiple genes coding for adenine nucleotide translocators which enables this intracellular parasite to exploit the cytoplasmic ATP of its host cell as a source of energy. The aim of this review is to describe the different aspects of the bioenergetic system in R. prowazekii and to discuss the results of phylogenetic reconstructions based on a variety of bioenergetic molecules which shed light on the origin and evolution of the mitochondrial genomes.

A phylogenetic analysis of the cytochrome b and cytochrome c oxidase I genes supports an origin of mitochondria from within the Rickettsiaceae.

We have cloned and sequenced the genes encoding cytochrome b (cob) and cytochrome c oxidase subunit I (cox1) from Rickettsia prowazekii, a member of the alpha-proteobacteria. The phylogenetic analysis supports the hypothesis that mitochondria are derived from the alpha-proteobacteria and more specifically from within the Rickettsiaceae. We have estimated that the common ancestor of mitochondria and Rickettsiaceae dates back to more than 1500 million years ago.

Obligate intracellular parasites: Rickettsia prowazekii and Chlamydia trachomatis.

Transitions to obligate intracellular parasitism have occurred at numerous times in the evolutionary past. The genome sequences of two obligate intracellular parasites, Rickettsia prowazekii and Chlamydia trachomatis, were published last year. A comparative analysis of these two genomes has revealed examples of reductive convergent evolution, such as a massive loss of genes involved in biosynthetic functions. In addition, both genomes were found to encode transport systems for ATP and ADP, not otherwise found in bacteria. Here, we discuss adaptations to intracellular habitats by comparing the information obtained from the recently published genome sequences of R. prowazekii and C. trachomatis.

Mutualists and parasites: how to paint yourself into a (metabolic) corner.

Eukaryotes have developed an elaborate series of interactions with bacteria that enter their bodies and/or cells. Genome evolution of symbiotic and parasitic bacteria multiplying inside eukaryotic cells results in both convergent and divergent changes. The genome sequences of the symbiotic bacteria of aphids, Buchnera aphidicola, and the parasitic bacteria of body louse and humans, Rickettsia prowazekii, provide insights into these processes. Convergent genome characteristics include reduction in genome sizes and lowered G+C content values. Divergent evolution was recorded for amino acid and cell wall biosynthetic genes. The presence of pseudogenes in both genomes provides examples of recent gene inactivation events and offers clues to the process of genome deterioration and host-cell adaptation.

Functional interactions between mutated forms of ribosomal proteins S4, S5 and S12.

Here we show that ram mutations, either in ribosomal protein S4 or S5, decrease the proofreading flows for both cognate and noncognate ternary complexes bound by streptomycin-dependent (SmD) ribosomes. This effect is accompanied by a slight increase in the overall error frequency. More important, however, is the decreased proofreading of the cognate species which is almost reduced to wild-type levels. The data suggest that it may be the reduction of the proofreading of the cognate substrate that is important for suppressing streptomycin dependence. Furthermore, we show that rpsE mutants, selected from streptomycin-dependent strains, behave kinetically very similarly to the previously described rpsD mutants.

A century of typhus, lice and Rickettsia.

At the beginning of the 20th century, it was discovered at the Pasteur Institute in Tunis that epidemic typhus is transmitted by the human body louse. The complete genome sequence of its causative agent, Rickettsia prowazekii, was determined at Uppsala University in Sweden at the end of the century. In this mini-review, we discuss insights gained from the genome sequence of this fascinating and deadly organism.

Multispacer typing technique for sequence-based typing of Bartonella quintana.

Bartonella quintana is a worldwide fastidious bacterium of the Alphaproteobacteria responsible for bacillary angiomatosis, trench fever, chronic lymphadenopathy, and culture-negative endocarditis. The recent genome sequencing of a B. quintana isolate allowed us to propose a genome-wide sequence-based typing method. To ensure sequence discrimination based on highly polymorphic areas, we amplified and sequenced 34 spacers in a large collection of B. quintana isolates. Six of these exhibited polymorphisms and allowed the characterization of 4 genotypes. However, the strain variants suggested by the noncoding sequences did not correlate with the results of pulsed-field gel electrophoresis (PFGE), which suggested a higher degree of variability. Modification of the PFGE profile of one isolate after nine subcultures confirmed that rearrangement frequencies are high in this species, making PFGE unreliable for epidemiological purposes. The low extent of sequence heterogeneity in the species suggests a recent emergence of this bacterium as a human pathogen. Direct typing of natural samples allowed the identification of a fifth genotype in the DNA extracted from a human body louse collected in Burundi. We have named the typing technique herein described multispacer typing.

Genomic evolution drives the evolution of the translation system.

Our thesis is that the characteristics of the translational machinery and its organization are selected in part by evolutionary pressure on genomic traits have nothing to do with translation per se. These genomic traits include size, composition, and architecture. To illustrate this point, we draw parallels between the structure of different genomes that have adapted to intracellular niches independently of each other. Our starting point is the general observation that the evolutionary history of organellar and parasitic bacteria have favored bantam genomes. Furthermore, we suggest that the constraints of the reductive mode of genomic evolution account for the divergence of the genetic code in mitochondria and the genetic organization of the translational system observed in parasitic bacteria. In particular, we associate codon reassignments in animal mitochondria with greatly simplified tRNA populations. Likewise, we relate the organization of translational genes in the obligate intracellular parasite Rickettsia prowazekii to the processes supporting the reductive mode of genomic evolution. Such findings provide strong support for the hypothesis that genomes of organelles and of parasitic bacteria have arisen from the much larger genomes of ancestral bacteria that have been reduced by intrachromosomal recombination and deletion events. A consequence of the reductive mode of genomic evolution is that the resulting translation systems may deviate markedly from conventional systems.

Genome degradation is an ongoing process in Rickettsia.

To study reductive evolutionary processes in bacterial genomes, we examine sequences in the Rickettsia genomes which are unconstrained by selection and evolve as pseudogenes, one of which is the metK gene, which codes for AdoMet synthetase. Here, we sequenced the metK gene and three surrounding genes in eight different species of the genus Rickettsia. The metK gene was found to contain a high incidence of deletions in six lineages, while the three genes in its surroundings were functionally conserved in all eight lineages. A more drastic example of gene degradation was identified in the metK downstream region, which contained an open reading frame in Rickettsia felis. Remnants of this open reading frame could be reconstructed in five additional species by eliminating sites of frameshift mutations and termination codons. A detailed examination of the two reconstructed genes revealed that deletions strongly predominate over insertions and that there is a strong transition bias for point mutations which is coupled to an excess of GC-to-AT substitutions. Since the molecular evolution of these inactive genes should reflect the rates and patterns of neutral mutations, our results strongly suggest that there is a high spontaneous rate of deletions as well as a strong mutation bias toward AT pairs in the Rickettsia genomes. This may explain the low genomic G + C content (29%), the small genome size (1.1 Mb), and the high noncoding content (24%), as well as the presence of several pseudogenes in the Rickettsia prowazekii genome.

Pseudogenes, junk DNA, and the dynamics of Rickettsia genomes.

Studies of neutrally evolving sequences suggest that differences in eukaryotic genome sizes result from different rates of DNA loss. However, very few pseudogenes have been identified in microbial species, and the processes whereby genes and genomes deteriorate in bacteria remain largely unresolved. The typhus-causing agent, Rickettsia prowazekii, is exceptional in that as much as 24% of its 1.1-Mb genome consists of noncoding DNA and pseudogenes. To test the hypothesis that the noncoding DNA in the R. prowazekii genome represents degraded remnants of ancestral genes, we systematically examined all of the identified pseudogenes and their flanking sequences in three additional Rickettsia species. Consistent with the hypothesis, we observe sequence similarities between genes and pseudogenes in one species and intergenic DNA in another species. We show that the frequencies and average sizes of deletions are larger than insertions in neutrally evolving pseudogene sequences. Our results suggest that inactivated genetic material in the Rickettsia genomes deteriorates spontaneously due to a mutation bias for deletions and that the noncoding sequences represent DNA in the final stages of this degenerative process.

GRS: a graphic tool for genome retrieval and segment analysis.

GRS is a graphic tool for retrieval and visualization of genome segments from partially or completely sequenced genomes. To facilitate visual identification of conserved genomic motifs, genes are color-coded according to their presumed functional roles. Aligned genes can be rapidly screened for potential homology by automatic retrieval and alignment of the corresponding protein sequences. Furthermore, the map location of any genome segment can be visually compared to the position of the same segment in other genomes or to the position of other segments within the same genome. The gene string analysis option of GRS allows the identification of genes that are identically arranged in any pairwise set of genomes. Finally, the program allows the user to create new gene table format files to enable comparisons of gene order structures in recently determined sequence data to the patterns of genes in already existing microbial and organellar databases. With the help of GRS, the genomic contexts of genes for which no identifiable homologues exist can be analyzed to provide an additional source of information for sequence annotations. We illustrate the use of GRS by analyzing the structure and distribution of phylogenetically conserved motifs in closely as well as more distantly related microbial genomes.

Codon usage and base composition in Rickettsia prowazekii.

Codon usage and base composition in sequences from the A + T-rich genome of Rickettsia prowazekii, a member of the alpha Proteobacteria, have been investigated. Synonymous codon usage patterns are roughly similar among genes, even though the data set includes genes expected to be expressed at very different levels, indicating that translational selection has been ineffective in this species. However, multivariate statistical analysis differentiates genes according to their G + C contents at the first two codon positions. To study this variation, we have compared the amino acid composition patterns of 21 R. prowazekii proteins with that of a homologous set of proteins from Escherichia coli. The analysis shows that individual genes have been affected by biased mutation rates to very different extents: genes encoding proteins highly conserved among other species being the least affected. Overall, protein coding and intergenic spacer regions have G + C content values of 32.5% and 21.4%, respectively. Extrapolation from these values suggests that R. prowazekii has around 800 genes and that 60-70% of the genome may be coding.

Effects of erythromycin base and erythromycin esters on protein synthesis in vivo in Escherichia coli.

The inhibitory effects on protein synthesis in vivo of erythromycin base and several erythromycin esters have been determined. An experimental method was used, especially suited for studies on short-lived antibiotics causing low levels of inhibition. Most of the tested derivatives were 2-3% as active as erythromycin base while a split product of erythromycin, anhydroerythromycin, was 4% as effective.