Charles G. Kurland

The genome sequence of Rickettsia prowazekii and the origin of mitochondria.

We describe here the complete genome sequence (1,111,523 base pairs) of the obligate intracellular parasite Rickettsia prowazekii, the causative agent of epidemic typhus. This genome contains 834 protein-coding genes. The functional profiles of these genes show similarities to those of mitochondrial genes: no genes required for anaerobic glycolysis are found in either R. prowazekii or mitochondrial genomes, but a complete set of genes encoding components of the tricarboxylic acid cycle and the respiratory-chain complex is found in R. prowazekii. In effect, ATP production in Rickettsia is the same as that in mitochondria. Many genes involved in the biosynthesis and regulation of biosynthesis of amino acids and nucleosides in free-living bacteria are absent from R. prowazekii and mitochondria. Such genes seem to have been replaced by homologues in the nuclear (host) genome. The R. prowazekii genome contains the highest proportion of non-coding DNA (24%) detected so far in a microbial genome. Such non-coding sequences may be degraded remnants of ‘neutralized’ genes that await elimination from the genome. Phylogenetic analyses indicate that R. prowazekii is more closely related to mitochondria than is any other microbe studied so far.

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.

Horizontal gene transfer: A critical view

It has been suggested that horizontal gene transfer (HGT) is the “essence of phylogeny.” In contrast, much data suggest that this is an exaggeration resulting in part from a reliance on inadequate methods to identify HGT events. In addition, the assumption that HGT is a ubiquitous influence throughout evolution is questionable. Instead, rampant global HGT is likely to have been relevant only to primitive genomes. In modern organisms we suggest that both the range and frequencies of HGT are constrained most often by selective barriers. As a consequence those HGT events that do occur most often have little influence on genome phylogeny. Although HGT does occur with important evolutionary consequences, classical Darwinian lineages seem to be the dominant mode of evolution for modern organisms.

Origin and Evolution of the Mitochondrial Proteome

SUMMARY 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 α-proteobacteria symbiont, proteins with no homology to bacterial orthologs, and diverse proteins with bacterial affinities not derived from α-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 α-proteobacterial endosymbiont is unclear, but there is no indication that it was an autotroph. There are no indications of a specific α-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.

Genomics and the Irreducible Nature of Eukaryote Cells

Large-scale comparative genomics in harness with proteomics has substantiated fundamental features of eukaryote cellular evolution. The evolutionary trajectory of modern eukaryotes is distinct from that of prokaryotes. Data from many sources give no direct evidence that eukaryotes evolved by genome fusion between archaea and bacteria. Comparative genomics shows that, under certain ecological settings, sequence loss and cellular simplification are common modes of evolution. Subcellular architecture of eukaryote cells is in part a physical-chemical consequence of molecular crowding; subcellular compartmentation with specialized proteomes is required for the efficient functioning of proteins.

Errors of heterologous protein expression.

Missense substitutions and processivity errors in the translation of heterologous proteins are expected to occur at higher frequencies than the corresponding errors of normal translation. The resulting error-containing products may overload chaperone systems. Likewise, there may be a risk of an immunogenic response to heterologous proteins introduced into vertebrates. Recent work has been carried out on the mechanisms by which such errors arise and on their occurrence in cloned, heterologous gene products.

Is there proofreading during polypeptide synthesis

The stoichiometric efficiency with which ternary complexes containing Phe‐tRNAphe and Leu‐tRNAleu support polypeptide synthesis has been compared in a poly(U)‐directed, steady‐state translation system. When unfractionated tRNA is used to support synthesis, the number of discharged ternary complexes per peptide bond formed is an average of 48 times greater for leucine than for phenylalanine. When three purified leucine isoacceptor species are tested, they each show a characteristic ratio of ternary complexes discharged per missense insertion, normalized to that for phenylalanine: these are 103, 76, and 45 for Leu‐ tRNA2leu, Leu‐ tRNA3leu, and Leu‐ tRNA4leu, respectively. The data are consistent with the functioning of a proofreading mechanism during translation.

Hyper-accurate ribosomes inhibit growth.

We have compared both in vivo and in vitro translation by ribosomes from wild‐type bacteria with those from streptomycin‐resistant (SmR), streptomycin‐dependent (SmD) and streptomycin‐pseudo‐dependent (SmP) mutants. The three mutant bacteria translate more accurately and more slowly in the absence of streptomycin (Sm) than do wild‐type bacteria. In particular, the SmP bacteria grow at roughly half the rate of the wild‐type in the absence of Sm. The antibiotic stimulates both the growth rate and the translation rate of SmP bacteria by approximately 2‐fold, but it simultaneously increases the nonsense suppression rate quite dramatically. Kinetic experiments in vitro show that the greater accuracy and slower translation rates of mutant ribosomes compared with wild‐type ribosomes are associated with much more rigorous proofreading activities of SmR, SmD and SmP ribosomes. Sm reduces the proofreading flows of the mutant ribosomes and stimulates their elongation rates. The data suggest that these excessively accurate ribosomes are kinetically less efficient than wild‐type ribosomes, and that this inhibits mutant growth rates. The stimulation of the growth of the mutants by Sm results from the enhanced translational efficiency due to the loss of proofreading, which more than offsets the loss of accuracy caused by the antibiotic.

Costs of accuracy determined by a maximal growth rate constraint.

The present study is best understood as an extension and critique of two schools of thought. The first is that of Malloe and his students, among whom we number ourselves. It is to Maaloe that we are indebted for the idea that logarithmically growing bacteria assemble and use tibosomes in amounts that are optimally adjusted to yield the maximal growth rates supported by different media. Her, we begin our analysis by applying this optimization priciple to all the components of a logarithmically growing system. Our objective is to use the growth optimization constraint as a tool to explore the physiological limits on the accuracy of gene expression. This brings us to our second source of inspiration, which is Orgels (1963) conception of a problem that Ninio (1982) has referred to as the ‘great error loop’.

The Dual Origin of the Yeast Mitochondrial Proteome

We propose a scheme for the origin of mitochondria based on phylogenetic reconstructions with more than 400 yeast nuclear genes that encode mitochondrial proteins. Half of the yeast mitochondrial proteins have no discernable bacterial homologues, while one‐tenth are unequivocally of α‐proteobacterial origin. These data suggest that the majority of genes encoding yeast mitochondrial proteins are descendants of two different genomic lineages that have evolved in different modes. First, the ancestral free‐living α‐proteobacterium evolved into an endosymbiont of an anaerobic host. Most of the ancestral bacterial genes were lost, but a small fraction of genes supporting bioenergetic and translational processes were retained and eventually transferred to what became the host nuclear genome. In a second, parallel mode, a larger number of novel mitochondrial genes were recruited from the nuclear genome to complement the remaining genes from the bacterial ancestor. These eukaryotic genes, which are primarily involved in transport and regulatory functions, transformed the endosymbiont into an ATP‐exporting organelle. Copyright