From stochastic environments to life histories and back

Environmental stochasticity is known to play an important role in life-history evolution, but most general theory assumes a constant environment. In this paper, we examine life-history evolution in a variable environment, by decomposing average individual fitness (measured by the long-run stochastic growth rate) into contributions from average vital rates and their temporal variation. We examine how generation time, demographic dispersion (measured by the dispersion of reproductive events across the lifespan), demographic resilience (measured by damping time), within-year variances in vital rates, within-year correlations between vital rates and between-year correlations in vital rates combine to determine average individual fitness of stylized life histories. In a fluctuating environment, we show that there is often a range of cohort generation times at which the fitness is at a maximum. Thus, we expect ‘optimal’ phenotypes in fluctuating environments to differ from optimal phenotypes in constant environments. We show that stochastic growth rates are strongly affected by demographic dispersion, even when deterministic growth rates are not, and that demographic dispersion also determines the response of life-history-specific average fitness to within- and between-year correlations. Serial correlations can have a strong effect on fitness, and, depending on the structure of the life history, may act to increase or decrease fitness. The approach we outline takes a useful first step in developing general life-history theory for non-constant environments.     

A fast algorithm for estimating transmission probabilities in QTL detection designs with dense maps

Background

In the case of an autosomal locus, four transmission events from the parents to progeny are possible, specified by the grand parental origin of the alleles inherited by this individual. Computing the probabilities of these transmission events is essential to perform QTL detection methods.

Results

A fast algorithm for the estimation of these probabilities conditional to parental phases has been developed. It is adapted to classical QTL detection designs applied to outbred populations, in particular to designs composed of half and/or full sib families. It assumes the absence of interference.

Conclusion

The theory is fully developed and an example is given.     

Visual Evidence of Horizontal Gene Transfer between Plants and Bacteria in the Phytosphere of Transplastomic Tobacco

Plant surfaces, colonized by numerous and diverse bacterial species, are often considered hot spots for horizontal gene transfer (HGT) between plants and bacteria. Plant DNA released during the degradation of plant tissues can persist and remain biologically active for significant periods of time, suggesting that soil or plant-associated bacteria could be in direct contact with plant DNA. In addition, nutrients released during the decaying process may provide a copiotrophic environment conducive for opportunistic microbial growth. Using Acinetobacter baylyi strain BD413 and transplastomic tobacco plants harboring the aadA gene as models, the objective of this study was to determine whether specific niches could be shown to foster bacterial growth on intact or decaying plant tissues, to develop a competence state, and to possibly acquire exogenous plant DNA by natural transformation. Visualization of HGT in situ was performed using A. baylyi strain BD413(rbcL-ΔPaadA::gfp) carrying a promoterless aadA::gfp fusion. Both antibiotic resistance and green fluorescence phenotypes were restored in recombinant bacterial cells after homologous recombination with transgenic plant DNA. Opportunistic growth occurred on decaying plant tissues, and a significant proportion of the bacteria developed a competence state. Quantification of transformants clearly supported the idea that the phytosphere constitutes a hot spot for HGT between plants and bacteria. The nondisruptive approach used to visualize transformants in situ provides new insights into environmental factors influencing HGT for plant tissues.Despite the annually increasing acreage planted with genetically modified plants worldwide, the ongoing debate on their ecological safety is controversial and gave impetus to different studies of the putative horizontal gene transfer (HGT) of recombinant DNA from plant to bacteria (12, 30). Research regarding the fate of plant transgenes in environmental microbial communities is driven by practical societal concerns related to the potential dissemination of antibiotic resistance determinants in the environment and by fundamental evolution questions about gene transfer between species and kingdoms. Different parts of a plant (globally defined as the phytosphere) support the growth of numerous and diverse bacteria that colonize the surfaces or internal tissues and display advantageous, neutral, or pathogenic functions toward the plant (1, 9, 22). However, the plant as a whole is exposed to many environmental challenges and does not always provide the same favorable conditions for bacterial growth. The latter depends on several factors, such as the presence of nutrients, moisture, shelter from desiccation and UV, and shelter from grazing and predation, all of which fluctuate rapidly and are heterogeneously distributed in and on the plant. Hence, bacterial growth seems to occur mostly in nutrient-rich, few, and localized microhabitats on plant surfaces where bacteria would form aggregates (17, 20, 21, 22, 33). The presence of large clusters of bacteria at sites of relative nutrient abundance on plant surfaces might also increase the potential for metabolic and genetic exchange (19). For example, bacterial growth and relatively high rates of transfer for a conjugative plasmid were reported to occur on plant surfaces (2, 3, 5). Similarly, availability of growth substrates, high bacterial density, and the presence of solid leaf surfaces were thought to induce gene transfer by conjugation in the phyllosphere at significantly high rates (26).Of the three mechanisms of bacterial HGT, natural transformation is considered to be the only one that could be effectively implicated in the transfer of DNA from transgenic plants to bacteria (4, 25). Although plants support bacterial growth, only putative evidence of DNA released by naturally degrading plant tissue being involved in a natural transformation process exists (23). Ceccherini et al. (6) showed, for example, that although most of the plant DNA was degraded within a short time by plant nucleases in planta during the process of plant decay, a measurable fraction escaped degradation and was still able to transform a recipient soil isolate in vitro (6).In order to assess plant-to-bacteria gene transfer, some studies have been conducted with different plant compartments. For example, the possibility for Acinetobacter baylyi strain BD413 to grow opportunistically in Ralstonia solanacearum-infected plant tissues revealed a new niche for this soil bacterium: the pathosphere. Moreover, this bacterium could be naturally transformed therein by artificially added or indigenous transgenic DNA (10, 11). Yet, other plant compartments could be as propitious to HGT; for example, the residuesphere (i.e., the naturally degrading plant material at the interface with soil) has been shown to provide conditions for growth and conjugal gene transfer between indigenous soil bacteria (7, 34). The litter and the residues of annual crops represent an important amount of final plant production, which are often left in the field after harvest and, in most cases, account for up to 60% of the world''s plant biomass (14).The assessment of natural transformation events in the soil or the phytosphere has, however, revealed several methodological challenges and biases, since quantification of transformation events has often been conducted with a cultivation-based approach requiring the plating of recipient bacteria on selective media supplemented with antibiotics. Antibiotic resistance determinants are widespread in soil environments, providing a technical intricacy in the discrimination between recipients with newly acquired traits and indigenous antibiotic-resistant flora with naturally fitted analogous genes. In addition, discrepancies between transformation frequencies determined on plates and those determined by cell densitometry revealed that the latter were usually higher by 2 orders of magnitude (37), leading to an underestimation of the phenomenon in natural settings. In addition, the uncertainty of whether each colony enumerated on a plate belongs to a single independent transformation event or is one of many clones (from one event) extracted from the sample makes transformation frequency calculations imprecise. Another drawback of the plating step is the disruptive sampling of material to rescue transformants, which averages the frequency calculations over the entire sample. Due to the spatial heterogeneity of available nutrients and biologically active DNA, localized spots, which are most conducive for HGT, might be delimited at microscale. However, to date, knowledge about the effective topology is lacking, although the heterogeneity of bacterial growth on plant surfaces has been shown (15, 20, 21, 22).The objectives of this study were to determine whether, during the natural or pathogen-induced decay of plant tissues, specific niches could be shown to foster bacterial growth, to develop a competence state, and to possibly acquire exogenous plant DNA using the Acinetobacter baylyi strain BD413 via natural transformation. Visualization of bacterial colonization plant material and detection of HGT events were performed at the leaf and bacterial scales using a cultivation-independent assay that relies upon a bioreporter tool (32). Microcosm-based experiments revealed that bacterial growth and competence development occur in different compartments of the plant. Isolation and direct visualization of transformants in situ suggest that some compartments of the phytosphere can be regarded as environmental hot spots for HGT.     

Impact of Therapeutic Treatment with β-Lactam on Transfer of the blaCTX-M-9 Resistance Gene from Salmonella enterica Serovar Virchow to Escherichia coli in Gnotobiotic Rats

The conjugative transfer of the plasmid carrying the bla_CTX-M-9 gene from Salmonella enterica serovar Virchow isolated from a chicken farm to a recipient Escherichia coli strain was evaluated in vitro and in axenic rats inoculated with both strains, with or without selective pressure due to therapeutic doses of cefixime. The transfer of the bla_CTX-M-9 gene of S. enterica serovar Virchow to E. coli was confirmed in vitro, at a low frequency of 5.9 × 10⁻⁸ transconjugants/donors. This transfer rate was higher in gnotobiotic rats and reached ∼10⁻⁵ transconjugants/donors without selective pressure. This frequency was not affected by the addition of therapeutic doses of cefixime. Thus, estimates of in vitro transfer underestimated potential transfer in the digestive tract, and therapeutic doses of cefixime did not increase the selection for transconjugants.β-Lactams and extended-spectrum cephalosporins are widely used in human and veterinary medicine to treat severe infections in humans and animals caused by Enterobacteriaceae and other gram-negative pathogens (7). However, extended-spectrum β-lactamases (ESBLs) have emerged and become the major mechanism of resistance to β-lactam antibiotics. Until the late 1990s, TEM and SHV enzymes were the predominant ESBLs. However, over the last decade, CTX-M-type enzymes have become the most prevalent extended-spectrum β-lactamases worldwide. Currently, there have been a number of reports documenting an increasing prevalence of enteric pathogens that produce these plasmid-mediated CTX-M enzymes (4, 8, 9, 39).Although originally confined to hospitals, ESBL-producing strains are now emerging in the community. Several investigations have shown that the rate of fecal carriage of ESBL-positive isolates (especially Escherichia coli) in humans is increasing, with CTX-M type enzymes found in most isolates (14, 17, 25, 28, 30, 36). This alarming phenomenon may have serious economic consequences and implications for treatment.bla_CTX-M genes spread throughout the community, mostly through the transmission of plasmids, and some studies have reported that animals may serve as a possible source for the dissemination of ESBL-encoding genes to humans. Indeed, common conjugative resistance plasmids and resistant clones have been found in animals, food products, and humans, suggesting that the transfer of extended-spectrum cephalosporin resistance between animals and humans is possible (17, 18, 21, 31, 40).In France, the emergence of the CTX-M-9 enzyme in Salmonella enterica serovar Virchow strains recovered from poultry, poultry products, and one human patient was reported between 2002 and 2003 (5, 31, 38, 40, 41). A comparative analysis of the bla_CTX-M-2 and bla_CTX-M-9 plasmids from S. enterica serovar Virchow isolates from human and poultry sources demonstrated a close relationship between the plasmids (16).To examine the hypothesis that antimicrobial resistance in humans could partly be attributed to food products, a number of studies have investigated the transfer of antibiotic resistance from bacteria originating in animal hosts or food products. Previously, Lester et al. (24) demonstrated the transfer of the vanA resistance gene from Enterococcus faecium isolated from animals to E. faecium isolated from human volunteers during transient intestinal colonization in humans in the absence of selective pressure. In a gnotobiotic mouse model, Feld et al. demonstrated the evidence of spread of a small plasmid pLFE1 harboring the erythromycin-resistant gene erm(B) from Lactobacillus plantarum isolated from raw-milk cheese to exogenous Enterococcus faecalis inoculated into the intestinal flora of mice (16). The transfer rate was enhanced when erythromycin was coadministered. Improvements in our understanding of the in vivo transmissibility and stability of antimicrobial resistance markers and the mechanisms driving horizontal transmission may help to predict the outcome of different strategies for controlling epidemic plasmids.The aim of the present study was to determine whether the bla_CTX-M-9 resistance gene could be transferred from an animal S. enterica serovar Virchow strain to a commensal E. coli strain that originated from the human intestinal tract while under selective pressure from a therapeutic dose of β-lactam. The transfer was investigated by using in vitro mating and a germfree rat model. We also evaluated the impact of the concentration of cefixime, an expanded-spectrum cephalosporin, on the transfer rate.     

Hepatitis C Virus NS5A Protein Is a Substrate for the Peptidyl-prolyl cis/trans Isomerase Activity of Cyclophilins A and B

We report here a biochemical and structural characterization of domain 2 of the nonstructural 5A protein (NS5A) from the JFH1 Hepatitis C virus strain and its interactions with cyclophilins A and B (CypA and CypB). Gel filtration chromatography, circular dichroism spectroscopy, and finally NMR spectroscopy all indicate the natively unfolded nature of this NS5A-D2 domain. Because mutations in this domain have been linked to cyclosporin A resistance, we used NMR spectroscopy to investigate potential interactions between NS5A-D2 and cellular CypA and CypB. We observed a direct molecular interaction between NS5A-D2 and both cyclophilins. The interaction surface on the cyclophilins corresponds to their active site, whereas on NS5A-D2, it proved to be distributed over the many proline residues of the domain. NMR heteronuclear exchange spectroscopy yielded direct evidence that many proline residues in NS5A-D2 form a valid substrate for the enzymatic peptidyl-prolyl cis/trans isomerase (PPIase) activity of CypA and CypB.Hepatitis C virus (HCV)⁴ is a small, positive strand, RNA-enveloped virus belonging to the Flaviviridae family and the genus Hepacivirus. With 120–180 million chronically infected individuals worldwide, hepatitis C virus infection represents a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (1). The HCV viral genome (∼9.6 kb) codes for a unique polyprotein of ∼3000 amino acids (recently reviewed in Refs. 2–4). Following processing via viral and cellular proteases, this polyprotein gives rise to at least 10 viral proteins, divided into structural (core, E1, and E2 envelope glycoproteins) and nonstructural proteins (p7, NS2, NS3, NS4A, NS4B, NS5A, NS5B). Nonstructural proteins are involved in polyprotein processing and viral replication. The set composed of NS3, NS4A, NS4B, NS5A, and NS5B constitutes the minimal protein component required for viral replication (5).Cyclophilins are cellular proteins that have been identified first as CsA-binding proteins (6). As FK506-binding proteins (FKBP) and parvulins, cyclophilins are peptidyl-prolyl cis/trans isomerases (PPIase) that catalyze the cis/trans isomerization of the peptide linkage preceding a proline (6, 7). Several subtypes of cyclophilins are present in mammalian cells (8). They share a high sequence homology and a well conserved three-dimensional structure but display significant differences in their primary cellular localization and in abundance (9). CypA, the most abundant of the cyclophilins, is primarily cytoplasmic, whereas CypB is directed to the endoplasmic reticulum lumen or the secretory pathway. CypD, on the other hand, is the mitochondrial cyclophilin. Cyclophilins are involved in numerous physiological processes such as protein folding, immune response, and apoptosis and also in the replication cycle of viruses including vaccinia virus, vesicular stomatitis virus, severe acute respiratory syndrome (SARS)-coronavirus, and human immunodeficiency virus (HIV) (for review see Ref. 10). For HIV, CypA has been shown to interact with the capsid domain of the HIV Gag precursor polyprotein (11). CypA thereby competes with capsid domain/TRIM5 interaction, resulting in a loss of the antiviral protective effect of the cellular restriction factor TRIM5α (12, 13). Moreover, it has been shown that CypA catalyzes the cis/trans isomerization of Gly²²¹-Pro²²² in the capsid domain and that it has functional consequences for HIV replication efficiency (14–16). For HCV, Watashi et al. (17) have described a molecular and functional interaction between NS5B, the viral RNA-dependent RNA polymerase (RdRp), and cyclophilin B (CypB). CypB may be a key regulator in HCV replication by modulating the affinity of NS5B for RNA. This regulation is abolished in the presence of cyclosporin A (CsA), an inhibitor of cyclophilins (6). These results provided for the first time a molecular mechanism for the early-on observed anti-HCV activity of CsA (18–20). Although this initial report suggests that only CypB would be involved in the HCV replication process (17), a growing number of studies have recently pointed out a role for other cyclophilins (21–25).In vitro selection of CsA-resistant HCV mutants indicated the importance of two HCV nonstructural proteins, NS5B and NS5A (26), with a preponderant effect for mutations in the C-terminal half of NS5A. NS5A is a large phosphoprotein (49 kDa), indispensable for HCV replication and particle assembly (27–29), but for which the exact function(s) in the HCV replication cycle remain to be elucidated. This nonstructural protein is anchored to the cytoplasmic leaflet of the endoplasmic reticulum membrane via an N-terminal amphipathic α-helix (residues 1–27) (30, 31). Its cytoplasmic sequence can be divided into three domains: D1 (residues 27–213), D2 (residues 250–342), and D3 (residues 356–447), all connected by low complexity sequences (32). D1, a zinc-binding domain, adopts a dimeric claw-shaped structure, which is proposed to interact with RNA (33, 34). NS5A-D2 is essential for HCV replication, whereas NS5A-D3 is a key determinant for virus infectious particle assembly (27, 35). NS5A-D2 and -D3, for which sequence conservation among HCV genotypes is significantly lower than for D1, have been proposed to be natively unfolded domains (28, 32). Molecular and structural characterization of NS5A-D2 from HCV genotype 1a has confirmed the disordered nature of this domain (36, 37).As it is still not clear which cyclophilins are cofactors for HCV replication, and as mutations in HCV NS5A protein have been associated with CsA resistance, we decided to examine the interaction between both CypA and CypB and domain 2 of the HCV NS5A protein. We first characterized, at the molecular level, NS5A-D2 from the HCV JFH1 infectious strain (genotype 2a) and showed by NMR spectroscopy that this natively unfolded domain indeed interacts with both cyclophilin A and cyclophilin B. Our NMR chemical shift mapping experiments indicated that the interaction occurs at the level of the cyclophilin active site, whereas it lacks a precise localization on NS5A-D2. A peptide derived from the only well conserved amino acid motif in NS5A-D2 did interact with cyclophilin A but only with a 10-fold lower affinity than the full domain. We concluded from this that the many proline residues form multiple anchoring points, especially when they adopt the cis conformation. NMR exchange spectroscopy further demonstrated that NS5A-D2 is a substrate for the PPIase activities of both CypA and CypB. Both the NS5A/cyclophilin interaction and the PPIase activity of the cyclophilins on NS5A-D2 were abolished by CsA, underscoring the specificity of the interaction.     

Investigation towards bivalent chemically defined glycoconjugate immunogens prepared from acid-detoxified lipopolysaccharide of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> O1, serotype Inaba

A free amino group present on the acid-detoxified lipopolysaccharide (pmLPS) of V. cholerae O1 serotype Inaba was investigated for site-specific conjugation. Chemoselective pmLPS biotinylation afforded the corresponding mono-functionalized derivative, which retained antigenicity. Thus, pmLPS was bound to carrier proteins using thioether conjugation chemistry. Induction of an anti-LPS antibody (Ab) response in BALB/c mice was observed for all conjugates. Interestingly, the sera had vibriocidal activity against both Ogawa and Inaba strains opening the way to a possible bivalent vaccine. However, the level of this Ab response was strongly affected by both the nature of the linker and of the carrier. Furthermore, no switch from IgM to IgG, i.e. from a T cell-independent to a T cell-dependent immune response was detected, a result tentatively explained by the possible presence of free polysaccharide in the formulation. Taken together, these results encourage further investigation towards the development of potent pmLPS-based neoglycoconjugate immunogens, fully aware of the challenge faced in the development of a cholera vaccine that will provide efficient serogroup coverage.     

Engineering an efficient secretion of leech carboxypeptidase inhibitor in Escherichia coli

Background  

Despite advances in expression technologies, the efficient production of heterologous secreted proteins in Escherichia coli remains a challenge. One frequent limitation relies on their inability to be exported to the E. coli periplasm. However, recent studies have suggested that translational kinetics and signal sequences act in concert to modulate the export process.     

Characterization of proADAMTS5 processing by proprotein convertases

ADAMTS5 (aggrecanase-2), a key metalloprotease mediating cartilage destruction in arthritis, is synthesized as a zymogen, proADAMTS5. We report a detailed characterization of the propeptide excision mechanism and demonstrate that it is a major regulatory step with unusual characteristics. Using furin-deficient cells and a furin inhibitor, we found that proADAMTS5 was processed by proprotein convertases, specifically furin and PC7, but not PC6B. Mutagenesis of three sites containing basic residues within the ADAMTS5 propeptide (RRR(46), RRR(69) and RRRRR(261)) suggested that proADAMTS5 processing occurs after Arg(261). That furin processing was essential for ADAMTS5 activity was illustrated using the known ADAMTS5 substrate aggrecan, as well as a new substrate, versican, an important regulatory proteoglycan during mammalian development. When compared to other ADAMTS proteases, proADAMTS5 processing has several distinct features. In contrast to ADAMTS1, whose furin processing products were clearly present intracellularly, cleaved ADAMTS5 propeptide and mature ADAMTS5 were found exclusively in the conditioned medium. Despite attempts to enhance detection of intracellular proADAMTS5 processing, such as by immunoprecipitation of total ADAMTS5, overexpression of furin, and secretion blockade by monensin, neither processed ADAMTS5 propeptide nor the mature enzyme were found intracellularly, which was strongly suggestive of extracellular processing. Extracellular ADAMTS5 processing was further supported by activation of proADAMTS5 added exogenously to HEK293 cells stably expressing furin. Unlike proADAMTS9, which is processed by furin at the cell-surface, to which it is bound, ADAMTS5 does not bind the cell-surface. Thus, the propeptide processing mechanism of ADAMTS5 has several points of distinction from those of other ADAMTS proteases, which may have considerable significance in the context of osteoarthritis.     

Dissecting the Unique Nucleotide Specificity of Mimivirus Nucleoside Diphosphate Kinase

The analysis of the Acanthamoeba polyphaga mimivirus genome revealed the first virus-encoded nucleoside diphosphate kinase (NDK), an enzyme that is central to the synthesis of RNA and DNA, ubiquitous in cellular organisms, and well conserved among the three domains of life. In contrast with the broad specificity of cellular NDKs for all types of ribo- and deoxyribonucleotides, the mimivirus enzyme exhibits a strongly preferential affinity for deoxypyrimidines. In order to elucidate the molecular basis of this unique substrate specificity, we determined the three-dimensional (3D) structure of the Acanthamoeba polyphaga mimivirus NDK alone and in complex with various nucleotides. As predicted from a sequence comparison with cellular NDKs, the 3D structure of the mimivirus enzyme exhibits a shorter Kpn loop, previously recognized as a main feature of the NDK active site. The structure of the viral enzyme in complex with various nucleotides also pinpointed two residue changes, both located near the active site and specific to the viral NDK, which could explain its stronger affinity for deoxynucleotides and pyrimidine nucleotides. The role of these residues was explored by building a set of viral NDK variants, assaying their enzymatic activities, and determining their 3D structures in complex with various nucleotides. A total of 26 crystallographic structures were determined at resolutions ranging from 2.8 Å to 1.5 Å. Our results suggest that the mimivirus enzyme progressively evolved from an ancestral NDK under the constraints of optimizing its efficiency for the replication of an AT-rich (73%) viral genome in a thymidine-limited host environment.Mimivirus, a DNA virus infecting Acanthamoeba, is the largest and most complex virus isolated to date (8, 37). It is the first representative and prototype member of the Mimiviridae, the latest addition to the large nucleocytoplasmic DNA viruses, including the poxviruses, the phycodnaviruses, (infecting algae), the iridoviruses (infecting invertebrates and fishes), and asfarvirus (the agent of a swine fever in Africa) (18). The mimivirus''s record genome size (1.2 Mb) and gene content (911 encoded proteins), as well as the presence of genes previously thought to be specific to cellular organisms (such as aminoacyl-tRNA synthetases [3]), revived the debate about the evolutionary origin of DNA viruses and their putative role in the emergence of the eukaryote nucleus (reviewed in reference 7) or in the advent of DNA genomes (13).In this peculiar context, we found the discovery of the first virus-encoded nucleoside diphosphate kinase (NDK) within the mimivirus genome of great interest and warranting a detailed study of the structural and biochemical properties of this unique viral enzyme. Ubiquitous in cellular organisms, NDKs are responsible for the last step of 2′-deoxynucleoside triphosphate (dNTP) pathways and as such play an essential role in the replication of DNA by providing the basic precursors for its synthesis. Acting indiscriminately on ribonucleotides and deoxyribonucleotides, the cellular NDKs are also responsible for supplying energy to various essential synthetic pathways, producing NTPs for RNA synthesis, CTP for lipid synthesis, UTP for polysaccharide synthesis, and GTP for protein synthesis elongation, signal transduction, and microtubules polymerization. Besides their direct role in the above metabolic pathways, cellular NDKs have been involved in the regulation of cell growth and differentiation in vertebrates (22).Cellular NDKs are small proteins of about 150 amino acids, the sequences of which are highly conserved among the three domains of life (>40% identity). They are most often hexameric enzymes, with a few occurrences of tetrameric and dimeric NDK structures in bacteria (19, 25, 26, 31, 38). They all catalyze the transfer of a phosphate group from an NTP onto a nucleotide diphosphate (NDP) through an Mg²⁺-dependent reaction. In vivo, the phosphate donor is usually the nonlimiting ATP nucleotide.In agreement with their implication in various metabolic pathways, cellular NDKs exhibit little substrate specificity and are equally able to act on purine and pyrimidine nucleotides, in their 2′ OH and deoxyribonucleotide forms. In clear contrast, our characterization of the mimivirus NDK revealed its enhanced affinity for deoxypyrimidine nucleotides (20). This marked difference between the viral and cellular NDKs offered a good opportunity to explore the sequence and structure features governing substrate specificity. For instance, cellular NDKs exhibit a conserved loop, the Kpn loop, involved both in substrate binding and in oligomerization of the enzyme (19). Interestingly, a sequence comparison predicted this loop to be shorter in the Acanthamoeba polyphaga mimivirus NDK (NDK_apm) sequence. However, many other single-residue changes could also be involved in modifying the enzyme properties. To explore these issues, we performed a detailed structure-function analysis of the NDK_apm protein in a variety of mutated forms and substrate-enzyme complexes. Despite its markedly different sequence, the three-dimensional structure of the mimivirus NDK was found to be very similar to that of cellular enzymes. Its peculiar substrate specificity is not attributable to a single sequence feature but rather appears to result from the conjunction of several factors, suggesting the progressive optimization of an ancestral enzyme for the replication of an AT-rich (73%) genome in a thymidine-limited host environment.