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Evolution of the genomic systems of prokaryotes and its momentous consequences.

The earliest self-reproducing cell on Earth, our common ancestor, was probably as small as present-day bacteria. It gave rise to a very large and durable clone whose descendants must have been the only living occupants of the oceans for about one thousand million years. They reached astronomical numbers of separate, disjunct cells, and synthesized many new genes. Their small volume could not accommodate ever larger genomes and useful new genes replaced resident, less successful sequences, thus increasing diversity and the number of strains with highly specialized, distinct, bioenergetic potentialities. Also, selective pressure favored strains able to participate successfully in division of labor and in the sharing of diverse abilities in mixed communities, counterbalancing the limited capacities of individual genomes. Lateral gene transfer mechanisms appeared and were progressively improved, furthering the development of diversity. The prokaryotes' constructive evolution resulted in the formation of a worldwide web of genetic information, and a global bacterial superbiosystem (superorganism). By contrast, eukaryotic evolution of organisms has been typically Darwinian. Diversification of eukaryotic organisms was, however, considerably enriched and accelerated by symbioses with prokaryotes. The more broadly diversified bioenergetic potential of prokaryotes considerably increased the diversity of eukaryotes. Without their participation, our biosphere would have remained much less diverse and less dynamic. Environmental homeostasis has been maintained all along by guided bacterial evolution.

Evolution, Molecular↗

Mobile genetic elements: the agents of open source evolution.

Horizontal genomics is a new field in prokaryotic biology that is focused on the analysis of DNA sequences in prokaryotic chromosomes that seem to have originated from other prokaryotes or eukaryotes. However, it is equally important to understand the agents that effect DNA movement: plasmids, bacteriophages and transposons. Although these agents occur in all prokaryotes, comprehensive genomics of the prokaryotic mobile gene pool or 'mobilome' lags behind other genomics initiatives owing to challenges that are distinct from cellular chromosomal analysis. Recent work shows promise of improved mobile genetic element (MGE) genomics and consequent opportunities to take advantage - and avoid the dangers - of these 'natural genetic engineers'. This review describes MGEs, their properties that are important in horizontal gene transfer, and current opportunities to advance MGE genomics.

Conjugation, Genetic↗

The evolution of mutator genes in bacterial populations: the roles of environmental change and timing.

Recent studies have found high frequencies of bacteria with increased genomic rates of mutation in both clinical and laboratory populations. These observations may seem surprising in light of earlier experimental and theoretical studies. Mutator genes (genes that elevate the genomic mutation rate) are likely to induce deleterious mutations and thus suffer an indirect selective disadvantage; at the same time, bacteria carrying them can increase in frequency only by generating beneficial mutations at other loci. When clones carrying mutator genes are rare, however, these beneficial mutations are far more likely to arise in members of the much larger nonmutator population. How then can mutators become prevalent? To address this question, we develop a model of the population dynamics of bacteria confronted with ever-changing environments. Using analytical and simulation procedures, we explore the process by which initially rare mutator alleles can rise in frequency. We demonstrate that subsequent to a shift in environmental conditions, there will be relatively long periods of time during which the mutator subpopulation can produce a beneficial mutation before the ancestral subpopulations are eliminated. If the beneficial mutation arises early enough, the overall frequency of mutators will climb to a point higher than when the process began. The probability of producing a subsequent beneficial mutation will then also increase. In this manner, mutators can increase in frequency over successive selective sweeps. We discuss the implications and predictions of these theoretical results in relation to antibiotic resistance and the evolution of mutation rates.

Bacteria↗

A bacterial conjugation machinery recruited for pathogenesis.

Type IV secretion systems (T4SS) are multicomponent transporters of Gram-negative bacteria adapted to functions as diverse as DNA transfer in bacterial conjugation or the delivery of effector proteins into eukaryotic target cells in pathogenesis. The generally modest sequence conservation between T4SS may reflect their evolutionary distance and/or functional divergence. Here, we show that the establishment of intraerythrocytic parasitism by Bartonella tribocorum requires a putative T4SS, which shares an unprecedented level of sequence identity with the Trw conjugation machinery of the broad-host-range antibiotic resistance plasmid R388 (up to 80% amino acid identity for individual T4SS components). The highly conserved T4SS loci are collinear except for the presence of numerous tandem gene duplications in B. tribocorum, which mostly encode variant forms of presumed surface-exposed pilus subunits. Conservation is not only structural, but also functional: R388 mutated in either trwD or trwH encoding essential T4SS components could be trans-complemented for conjugation by the homologues of the B. tribocorum system. Conservation also includes the transcription regulatory circuit: both T4SS loci encode a highly homologous and interchangeable KorA/KorB repressor system that negatively regulates the expression of all T4SS components. This striking example of adaptive evolution reveals the capacity of T4SS to assume dedicated functions in either DNA transfer or pathogenesis over rather short evolutionary distance and implies a novel role for the conjugation systems of widespread broad-host-range plasmids in the evolution of bacterial pathogens.

Bartonella↗

Molecular evolution of Mycobacterium tuberculosis: phylogenetic reconstruction of clonal expansion.

SETTING: M. tuberculosis isolates were collected from patients attending health clinics in a high incidence urban community and in a low incidence rural setting in South Africa. OBJECTIVE: To reconstruct the evolutionary history of a group of closely related M. tuberculosis isolates using IS6110, DRr and MTB484(1) restriction fragment length polymorphism (RFLP) data. DESIGN: Mycobacterium tuberculosis isolates containing an average of ten IS6110 elements, with a similarity index of > or = 65% were genotypically classified by DNA fingerprinting using the IS6110 derived probes IS-3' and IS-5', as well as the DRr and MTB484(1) probes, in combination with PvuII or Hinfl endonuclease digestion. These RFLP data were subjected to phylogenetic analysis using both genetic distance and parsimony algorithms. RESULTS: Phylogenetic analysis predicted the existence of two independently evolving lineages, possibly evolving from a common ancestral strain. The topology of the phylogenetic tree was supported by comprehensive bootstrapping and the specific partitioning of DNA methylation phenotypes. The observed difference in the branch lengths of the two lineages may suggest differential evolutionary rates. Isolates collected from different geographical regions demonstrate independent evolution, suggesting that it is highly unlikely that strains have been recently transmitted between the two regions. The number of evolutionary events identified in this strain family differs significantly from that of previously characterized strain families, implying that evolutionary rate may be strain family dependent. CONCLUSION: Based on this analysis we propose that the algorithm used to calculate recent epidemiological events should be revised to incorporate the evolutionary characteristics of individual strain families, thereby enhancing the accuracy of molecular epidemiological calculations.

Algorithms↗

Evolution of resistance patterns and identification of risk factors for Streptococcus pneumoniae colonisation in daycare centre attendees in Athens, Greece.

Two cross-sectional surveillance studies were conducted during the winters of 2000 and 2003 in Athens, Greece, to obtain nasopharyngeal swabs from healthy pre-school children attending kindergartens. A total of 460 strains were examined in 2000 and 485 strains in 2003, with carriage rates of 31.7% and 34.6%, respectively. Susceptibility patterns were evaluated for penicillin G, erythromycin, ceftriaxone, moxifloxacin, linezolid and telithromycin. Penicillin non-susceptibility increased from 20% to 34.9%, whereas erythromycin non-susceptibility increased from 23% to 30.5%. Resistance to both agents climbed from 7.5% to 22.3% (P<0.001). No isolates were found to be resistant to any of the other antimicrobial agents. Risk factors for carriage and/or antimicrobial resistance were also assessed.

Anti-Bacterial Agents↗

Evolution of DNA uptake signal sequences.

The DNA of some naturally competent species of bacteria contains a large number of evenly distributed copies of a short sequence. This highly overrepresented sequence is believed to be an uptake signal sequence (USS) that helps bacteria to take up DNA selectively from (dead) members of their own species. For some time it has been assumed that the USS evolved in order to enable bacteria to distinguish between conspecific and nonconspecific DNA fragments (the preference-first hypothesis). Recently, Redfield suggested that this hypothesis is not in fact realistic, as it would require biologically implausible group selection. In this article we present a model designed to demonstrate the emergence of similar USSs in a population of simulated evolving agents. We use this model to examine the conditions under which a USS will emerge in a preference-first scenario.

Bacteria↗

Mycobacterium tuberculosis molecular evolution in western Mediterranean Islands of Sicily and Sardinia.

In this study, a total of 204 Mycobacterium tuberculosis DNAs from Sicily (n = 144) and Sardinia (n = 60) were studied by three genotyping methods. Results were analyzed both within and across islands, to define the phylogeographical specificities of the genotypes, look for their diversity and infer a molecular evolutionary scenario. A strong link between geography and tuberculosis genotypes was observed in Sardinia. The results were also matched against a world-wide genetic diversity database to compare the population structure of the tubercle bacilli in the islands. Eight common genotypes between Sicily, Sardinia and continental Italy were found which underlines the influences of the Italian mainland on the population structure on the islands and vice versa. A unified evolutionary scenario of M. tuberculosis evolution was built using numerical taxonomy and maximum parsimony (MP) methods. The finding of multiple families of M. tuberculosis strains (S, T, LAM, Haarlem), their presumed links with the major genetic groups (MGG) of M. tuberculosis complex, supports the view of independent introduction of several ancestral genotypes in Sicily and in Sardinia. We conclude that the two PCR-based genotyping combination (spoligotyping-VNTR) is an excellent tool to reconstruct M. tuberculosis phylogeny, that may be used to construct global and local evolutionary scenarios of the M. tuberculosis complex. The results obtained are paradigmatic of the complex interplay that exists between epidemic dynamics and evolutionary genetics of M. tuberculosis.

DNA, Bacterial↗

Comparative genomics reveals expansion of the FLC region in the genus Arabidopsis.

Mechanisms of genome evolution are poorly understood although recent genome sequencing is providing the tools to begin to illuminate such mechanisms. Using high-resolution molecular cytogenetic tools, we examined the structural evolution of 790 kb surrounding the evolutionarily important FLC locus of Arabidopsis thaliana in three of its relatives, Arabidopsis halleri, Arabidopsis neglecta and Arabidopsis arenosa. Sequenced BACs from A. thaliana were used as heterologous probes across these species and genome expansion was found in all three species relative to A. thaliana, ranging from 16 to 27%. Expansion was seen along the length of the entire region but molecular analyses revealed no characteristic pattern of either intra- or intergenic expansion among these species. Mapping of BACs on DNA fibers from A. thaliana revealed one possible error, approximately 14 kb missing from the reported sequence, indicating that for comparative studies it is important to confirm the reference sequence to which comparison will be made.

Arabidopsis↗

Evolution and microsynteny of the apyrase gene family in three legume genomes.

Apyrases have been suggested to play important roles in plant nutrition, photomorphogenesis, and nodulation. To help trace the evolution of these genes in the legumes--and possibly, the acquisition of new functions for nodulation--apyrase-containing BACs were sequenced from three legume genomes. Genomic sequences from Medicago truncatula, Glycine max and Lotus japonicus were compared to one another and to corresponding regions in Arabidopsis thaliana. A phylogenetic analysis of apyrase homologs from these regions and sequences from other legume species, as well as other plant families, identified a potentially legume-specific clade that contains a well-characterized soybean ( G. soja) apyrase, Gs52, as well as homologs from Dolichos, Lotus, Medicago and Pisum. Sister clades contain homologs from members of Brassicaceae, Solanaceae, Poaceae and Fabaceae. Comparisons of rates of change at synonymous and nonsynonymous sites in the Gs52 and sister clades show rapid evolution in the potentially legume-specific Gs52 clade. The genomic organization of the apyrase-containing BACs shows evidence of gene duplication, genomic rearrangement, and gene conversion among Gs52 homologs. Taken together, these results suggest a scenario of local apyrase gene duplication in an ancestor of the legumes, followed by functional diversification and increased rates of change in the new genes, and further duplications in the Galegae (which include the genera Medicago and Pisum). The study also provides a detailed comparison of genomic regions between two model genomes which are now being sequenced ( M. truncatulaand L. japonicus), and a genome from an economically important legume species ( G. max).

Apyrase↗

Utilization of a zebra finch BAC library to determine the structure of an avian androgen receptor genomic region.

The zebra finch (Taeniopygia guttata) is an important model organism for studying behavior, neuroscience, avian biology, and evolution. To support the study of its genome, we constructed a BAC library (TG__Ba) using DNA from livers of females. The BAC library consists of 147,456 clones with 98% containing inserts of an average size of 134 kb and represents 15.5 haploid genome equivalents. By sequencing a whole BAC, a full-length androgen receptor open reading frame was identified, the first in an avian species. Comparison of BAC end sequences and the whole BAC sequence with the chicken genome draft sequence showed a high degree of conserved synteny between the zebra finch and the chicken genome.

Animals↗

Silent nucleotide polymorphisms and a phylogeny for Mycobacterium tuberculosis.

Much remains unknown of the phylogeny and evolution of Mycobacterium tuberculosis, an organism that kills 2 million people annually. Using a population-based approach that analyzes multiple loci around the chromosome, we demonstrate that neutral genetic variation in genes associated with antimicrobial drug resistance has sufficient variation to construct a robust phylogenetic tree for M. tuberculosis. The data describe a clonal population with a minimum of four distinct M. tuberculosis lineages, closely related to M. bovis. The lineages are strongly geographically associated. Nucleotide substitutions proven to cause drug resistance are distributed throughout the tree, whereas nonsynonymous base substitutions unrelated to drug resistance have a restricted distribution. The phylogenetic structure is concordant with all the previously described genotypic and phenotypic groupings of M. tuberculosis strains and provides a unifying framework for both epidemiologic and evolutionary analysis of M. tuberculosis populations.

Amidohydrolases↗

Mercury adaptation among bacteria from a deep-sea hydrothermal vent.

Since deep-sea hydrothermal vent fluids are enriched with toxic metals, it was hypothesized that (i) the biota in the vicinity of a vent is adapted to life in the presence of toxic metals and (ii) metal toxicity is modulated by the steep physical-chemical gradients that occur when anoxic, hot fluids are mixed with cold oxygenated seawater. We collected bacterial biomass at different distances from a diffuse flow vent at 9 degrees N on the East Pacific Rise and tested these hypotheses by examining the effect of mercuric mercury [Hg(II)] on vent bacteria. Four of six moderate thermophiles, most of which were vent isolates belonging to the genus Alcanivorax, and six of eight mesophiles from the vent plume were resistant to >10 microM Hg(II) and reduced it to elemental mercury [Hg(0)]. However, four psychrophiles that were isolated from a nearby inactive sulfide structure were Hg(II) sensitive. A neighbor-joining tree constructed from the deduced amino acids of a PCR-amplified fragment of merA, the gene encoding the mercuric reductase (MR), showed that sequences obtained from the vent moderate thermophiles formed a unique cluster (bootstrap value, 100) in the MR phylogenetic tree, which expanded the known diversity of this locus. The temperature optimum for Hg(II) reduction by resting cells and MR activity in crude cell extracts of a vent moderate thermophile corresponded to its optimal growth temperature, 45 degrees C. However, the optimal temperature for activity of the MR encoded by transposon Tn501 was found to be 55 to 65 degrees C, suggesting that, in spite of its original isolation from a mesophile, this MR is a thermophilic enzyme that may represent a relic of early evolution in high-temperature environments. Results showing that there is enrichment of Hg(II) resistance among vent bacteria suggest that these bacteria have an ecological role in mercury detoxification in the vent environment and, together with the thermophilicity of MR, point to geothermal environments as a likely niche for the evolution of bacterial mercury resistance.

Adaptation, Physiological↗

Towards integration of multiple alignment and phylogenetic tree construction.

A central problem in the study of molecular evolution is the reconstruction of the history of a set of biological sequences in the form of a phylogenetic tree. One step in calculating this tree is the computation of a multiple alignment. Most existing approaches treat the two problems of multiple alignment and tree construction as separate while in fact they influence each other. Based on three-way alignments of pre-aligned groups of sequences we adapt a commonly used tree construction procedure to produce both tree and multiple alignment simultaneously. In contrast to existing iterative algorithms the new method can change alignments made early in the course of the computation at a later stage. A sufficient criterion to prevent the introduction of edges with negative length reduces the number of three-way alignments that need to be computed. Applications of the new approach to the alignment of protein and of nucleic acid sequences are presented.

Amino Acid Sequence↗

The role of recombination and mutation in 16S-23S rDNA spacer rearrangements.

The intragenomic heterogeneity of the bacterial intergenic (16S-23S rDNA) spacer region (ISR) was analysed from the following species in which sequences for the complete rRNA operon (rrn) set have been determined (rrn number): Enterococcus faecalis (6) and E. faecium (6), Bacillus subtilis (10), Staphylococcus aureus (9), Vibrio cholerae (4), Haemophilus influenzae (6) and Escherichia coli (7). It was found that some spacer sequence blocks were highly conserved between operons of a genome, whereas the presence of others was variable. When these variations were analysed using the program PLATO and partial likelihood phylogenies determined by DNAml for each operon set, three regions showed significant (Z>3.3) spatial variation [Region I was 78-184 nt long (2.1 4.4) possibly due to recombination or selection. Within Region I, there was sequence block variation in all operon sets [some operons contained tRNA genes (tRNAala, tRNAile or tRNAglu), whereas others had sequence blocks such as VS2 (S. aureus) or rsl (E. coli)]. Q Analysis of the ISR sequence from E. faecalis and E. faecium showed that there was more interspecies than intraspecies variation (both in DNA sequence and in the presence or absence of blocks). Dot matrix analysis of the sequence blocks in the nine rrn ISRs from S. aureus showed that there was significant homology between VS2 and VS5/VS6. Furthermore, repeat motifs with only A or T were present in higher copy numbers in VS5/VS6 than in VS2. Since these sequence blocks (VS2 and VS5-VS6) are related, intragenic evolution resulting in AT expansion may have occurred between these two regions. A model is proposed that postulates a role for recombination and AT-expansion in intra-genomic ISR variations. This process may represent a general mechanism of concerted evolution for bacterial ISR rearrangements.

Bacteria↗

Diversity and evolution of hydrogenase systems in rhizobia.

Uptake hydrogenases allow rhizobia to recycle the hydrogen generated in the nitrogen fixation process within the legume nodule. Hydrogenase (hup) systems in Bradyrhizobium japonicum and Rhizobium leguminosarum bv. viciae show highly conserved sequence and gene organization, but important differences exist in regulation and in the presence of specific genes. We have undertaken the characterization of hup gene clusters from Bradyrhizobium sp. (Lupinus), Bradyrhizobium sp. (Vigna), and Rhizobium tropici and Azorhizobium caulinodans strains with the aim of defining the extent of diversity in hup gene composition and regulation in endosymbiotic bacteria. Genomic DNA hybridizations using hupS, hupE, hupUV, hypB, and hoxA probes showed a diversity of intraspecific hup profiles within Bradyrhizobium sp. (Lupinus) and Bradyrhizobium sp. (Vigna) strains and homogeneous intraspecific patterns within R. tropici and A. caulinodans strains. The analysis also revealed differences regarding the possession of hydrogenase regulatory genes. Phylogenetic analyses using partial sequences of hupS and hupL clustered R. leguminosarum and R. tropici hup sequences together with those from B. japonicum and Bradyrhizobium sp. (Lupinus) strains, suggesting a common origin. In contrast, Bradyrhizobium sp. (Vigna) hup sequences diverged from the rest of rhizobial sequences, which might indicate that those organisms have evolved independently and possibly have acquired the sequences by horizontal transfer from an unidentified source.

Bacterial Proteins↗

Complex patterns of copy number variation at sites of segmental duplications: an important category of structural variation in the human genome.

The structural diversity of the human genome is much higher than previously assumed although its full extent remains unknown. To investigate the association between segmental duplications that display constitutive copy number differences (CNDs) between humans and the great apes and those which exhibit polymorphic copy number variations (CNVs) between humans, we analysed a BAC array enriched with segmental duplications displaying such CNDs. This study documents for the first time that in addition to human-specific gains common to all humans, these duplication clusters (DCs) also exhibit polymorphic CNVs > 40 kb. Segmental duplication is known to have been a frequent event during human genome evolution. Importantly, among the CNV-associated genes identified here, those involved in transcriptional regulation were found to be significantly overrepresented. Complex patterns of variation were evident at sites of DCs, manifesting as inter-individual differentially sized copy number alterations at the same genomic loci. Thus, CNVs associated with segmental duplications do not simply represent insertion/deletion polymorphisms, but rather constitute a wide variety of rearrangements involving differential amplification and partial gains and losses with high inter-individual variability. Although the number of CNVs was not found to differ between Africans and Caucasians/Asians, the average number of variant patterns per locus was significantly lower in Africans. Thus, complex variation patterns characterizing segmental duplications result from relatively recent genomic rearrangements. The high number of these rearrangements, some of which are potentially recurrent, together with differences in population size and expansion dynamics, may account for the greater diversity of CNV in Caucasians/Asians as compared with Africans.

Animals↗

Genetic divergence of the rhesus macaque major histocompatibility complex.

The major histocompatibility complex (MHC) is comprised of the class I, class II, and class III regions, including the MHC class I and class II genes that play a primary role in the immune response and serve as an important model in studies of primate evolution. Although nonhuman primates contribute significantly to comparative human studies, relatively little is known about the genetic diversity and genomics underlying nonhuman primate immunity. To address this issue, we sequenced a complete rhesus macaque MHC spanning over 5.3 Mb, and obtained an additional 2.3 Mb from a second haplotype, including class II and portions of class I and class III. A major expansion of from six class I genes in humans to as many as 22 active MHC class I genes in rhesus and levels of sequence divergence some 10-fold higher than a similar human comparison were found, averaging from 2% to 6% throughout extended portions of class I and class II. These data pose new interpretations of the evolutionary constraints operating between MHC diversity and T-cell selection by contrasting with models predicting an optimal number of antigen presenting genes. For the clinical model, these data and derivative genetic tools can be implemented in ongoing genetic and disease studies that involve the rhesus macaque.

Animals↗