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A genome-wide in vivo screen reveals fitness pathways required for streptococcal infective endocarditis.

Infective endocarditis (IE) is a life-threatening disease most often caused by blood-borne bacteria that infect previously damaged cardiac tissue. Despite the importance of this disease, the genetic basis for IE-associated fitness remains poorly defined. Here, we present the first genome-wide in vivo analysis of bacterial fitness in a vertebrate model of IE. We identified 146 genes in Streptococcus sanguinis required for IE fitness, the majority of which had not previously been linked to endocarditis. These determinants cluster into conserved metabolic, cell envelope, transport, and regulatory pathways, representing a vast reservoir of potential targets for novel antimicrobial intervention. A subset of these genes was examined in Streptococcus mutans; all were found to be essential for IE fitness in this distantly related oral species as well, suggesting broad conservation. Using experimental evolution, we further show that disruption of key fitness pathways triggers reproducible compensatory "bypass" mechanisms. Together, these findings provide a comprehensive, genome-wide map of the bacterial niche-requirements for streptococcal infective endocarditis.

Animals

[Comparative analysis of blood coagulation and various indicators of microcirculation in patients with coronary arteriosclerosis and hypertension in the age aspect].

In patients with hypertensive disease and coronary atherosclerosis the blood-clotting potential increases on account of a rise in the level of procoagulants and inhibition of fibrinolysis. Age-specific differences in the characteristics of the coagulation and fibrinolytic system of the blood are levelled out parallel with the development of atherosclerosis and progressive evolution of hypertensive disease. In patients of advanced age a tendency toward a compensatory increased activity of erythrocytic anticoagulation factors is noted, finding its expression in a drop of the procoagulants level, and increase of anticoagulants and in a rising fibrinolytic activity. Changes in the state of the conjunctival microcirculation and the retinal hemodynamics correlate with the intensity of the pathological process and shifts in the blood coagulation system.

Adaptation, Physiological

Complete sequences of the rRNA genes of Drosophila melanogaster.

In this, the first of three papers, we present the sequence of the ribosomal RNA (rRNA) genes of Drosophila melanogaster. The gene regions of D. melanogaster rDNA encode four individual rRNAs: 18S (1,995 nt), 5.8S (123 nt), 2S (30 nt), and 28S (3,945 nt). The ribosomal DNA (rDNA) repeat of D. melanogaster is AT rich (65.9% overall), with the spacers being particularly AT rich. Analysis of DNA simplicity reveals that, in contrast to the intergenic spacer (IGS) and the external transcribed spacer (ETS), most of the rRNA gene regions have been refractory to the action of slippage-like events, with the exception of the 28S rRNA gene expansion segments. It would seem that the 28S rRNA can accommodate the products of slippage-like events without loss of activity. In the following two papers we analyze the effects of sequence divergence on the evolution of (1) the 28S gene "expansion segments" and (2) the 28S and 18S rRNA secondary structures among eukaryotic species, respectively. Our detailed analyses reveal, in addition to unequal crossing-over, (1) the involvement of slippage and biased mutation in the evolution of the rDNA multigene family and (2) the molecular coevolution of both expansion segments and the nucleotides involved with compensatory changes required to maintain secondary structures of RNA.

Animals

Site-directed mutagenesis of arginine 179 of thymidylate synthase. A nonessential substrate-binding residue.

X-ray structural studies have shown that Arg-179 of thymidylate synthase is complexed to bound inorganic phosphate or to the 5'-phosphate of the bound substrate dUMP. The importance of Arg-179 to the structure/function of thymidylate synthase is also indicated by its complete conservation among the 17 thymidylate synthases thus far sequenced. In the present work, Arg-179 has been replaced by Thr, Ala, Lys, and Glu using site-directed mutagenesis with a mixture of four synthetic oligonucleotides as primers. The mutant proteins complement thymidylate synthase-deficient Escherichia coli and show high enzyme activity. Each of these mutants has been purified to homogeneity, partially sequenced to verify the mutation, and has had its steady state kinetic parameters determined. The most significant effect of all mutations is localized to a decrease in the net rate of association of thymidylate synthase with dUMP; the Lys mutant also shows an apparent increase in the dissociation constant of the folate cofactor of the reaction. The high activity in the mutant enzymes is explained by "plasticity" of the enzyme and compensatory actions of the other Arg residues. Why the Arg-179 residue has been conserved during evolution remains an open question.

Amino Acid Sequence

Fitness of RNA virus decreased by Muller's ratchet.

Why sex exists remains an unsolved problem in biology. If mutations are on the average deleterious, a high mutation rate can account for the evolution of sex. One form of this mutational hypothesis is Muller's ratchet. If the mutation rate is high, mutation-free individuals become rare and they can be lost by genetic drift in small populations. In asexual populations, as Muller noted, the loss is irreversible and the load of deleterious mutations increases in a ratchet-like manner with the successive loss of the least-mutated individuals. Sex can be advantageous because it increases the fitness of sexual populations by re-creating mutation-free individuals from mutated individuals and stops (or slows) Muller's ratchet. Although Muller's ratchet is an appealing hypothesis, it has been investigated and documented experimentally in only one group of organisms--ciliated protozoa. I initiated a study to examine the role of Muller's ratchet on the evolution of sex in RNA viruses and report here a significant decrease in fitness due to Muller's ratchet in 20 lineages of the RNA bacteriophage phi 6. These results show that deleterious mutations are generated at a sufficiently high rate to advance Muller's ratchet in an RNA virus and that beneficial, backward and compensatory mutations cannot stop the ratchet in the observed range of fitness decrease.

Bacteriophages

Observing development through evolutionary eyes: a practical approach.

An argument is made that only through a detailed comparison of mutational mechanisms underlying the evolution of the genetic systems governing development, can the 'logic' of individual development be fully comprehended. To do this, it is essential to choose two or more genes (or their products) that interact in the establishment of a given function, and to compare the molecular basis of that interaction in closely related species. The rationale to this approach arises from observations of molecular co-evolution between interacting partners involved with given functions which have led to species specificity in the manner in which such functions are effected. Molecular coevolution reveals that divergence in sequence can be tolerated whilst biological functions are maintained, not because it is neutral and dispensable but because successful, compensatory changes can evolve in eukaryotic genomes that are in continuous states of flux.

Animals

Miniaturization, genome size and the origin of functional constraints in the visual system of salamanders.

During their evolution, many species of lungless salamanders (fam. Plethodontidae) have experienced a great increase in genome size and consequently in the size of their cells, including sensory receptors and neurons. In addition, some have become extremely miniaturized. The consequences of these events and the morphological compensatory processes are studied in the visual system of juvenile and adult salamanders.

Animals

Masticatory function and post-Pleistocene evolution in Nubia.

The present research focuses on craniofacial variation in Nubia over approximately 10,000 years. Samples were grouped according to their temporal location and subsistence pattern, and represent a transition from a hunting-gathering adaptation (Mesolithic) to a transitional hunting-gathering-agricultural adaptation (A-C Group) and finally to a fully agricultural adaptation (Meroitic/X-Group/Christian). The purposes were: (1) to compare the Mesolithic sample with the later Nubian populations; and (2) to evaluate further the hypothesis that change in Nubian craniofacial morphology was due to changing functional demands associated with the progressive change in subsistence adaptation and associated behavior. The results tend to support recent views that the Nubian Mesolithic population is probably ancestral to later Nubian groups, and that the masticatory-functional hypothesis can best account for craniofacial change among the Nubians since 12,000 B.P. According to this hypothesis systematic reduction in functional demand placed on the masticatory complex from the Mesolithic led, secondarily, to an alteration of the growth of the maxillomandibular complex such that the face became progressively less robust and more inferoposteriorly located relative to the cranial vault. Both the increase in the height of the vault relative to its length, producing a more "globular" appearance, and the reduction in dental size were tertiary, compensatory responses to altered facial size and position.

Biological Evolution

Pressor responsiveness to endothelin is not attenuated in gravid rats.

The aim of this study was to evaluate the effect of synthetic human/porcine endothelin (ET-1) on mean arterial blood pressure (MAP) in pregnant and non-pregnant rats and to compare this to the effects of two well characterized agonists, angiotensin II (AII), and vasopressin (VP). On day 14 of gestation (parturition day 22) polyethylene catheters were chronically implanted in the abdominal aorta for monitoring of MAP and in the vena cava for administration of drugs. Pressor responsiveness was measured in conscious freely moving animals on day 20 and again on the 7th day post-partum. All three agonists increased MAP in a dose related manner. However, whereas the sensitivity of pregnant rats (P) to AII and VP was significantly blunted compared to postpartal (PP) measurements, the MAP responses to ET-1 were the same in both groups. Moreover, the combined administration of ET-1 at a subpressor dose (0.05 pmol/100 g bw) and AII or VP at effective doses significantly potentiated (particularly in P) the pressor effects of AII and VP. These results demonstrate that ET-1 and possibly other vasoactive substances of endothelial origin, override the compensatory mechanism of normal pregnancy with respect to the blunted responsiveness to AII and VP. Such a mechanism may be of particular relevance in the evolution of pregnancy-induced hypertension.

Angiotensin II

Alteration of hepatocytes by subcarcinogenic exposure to n-2-fluorenylacetamide.

These experiments examined the effects of a single, subcarcinogenic dose of dimethylnitrosamine or N-hydroxyfluorenylacetamide when administered after a subcarcinogenic dietary regimen of N-2-fluorenylacetamide. Control rats that received either carcinogen diet alone or a single dose of carcinogen demonstrated neither hepatic nodules nor hepatocellular carcinomas. Those animals that received dimethylnitrosamine subsequent to carcinogen diet demonstrated many persistent hepatic nodules and 100% hepato-cellular carcinomas. These data support the concept that the nodules produced by subcarcinogenic ingestion of N-2-fluorenylacetamide are not composed simply of normal hepatocytes undergoing compensatory regeneration but consist of cells that have been altered by the carcinogen. One manifestation of this alteration is an increased susceptibility to further carcinogenic evolution.

Animals

Secondary structure as a constraint on the evolution of a plant viral satellite RNA.

The genetic variability and evolution of the satellite RNA (satRNA) of cucumber mosaic virus (CMV) was analyzed. Twenty-five CMV-satRNAs compared clustered into three main groups, and no correlation was found between genetic proximity and other characteristics (pathogenicity, geographical origin) of the satRNAs. Values for the number of nucleotide substitutions per site between any two satRNAs suggest that divergence is checked by functional constraints. The analysis of mutations relative to an ancestral sequence, and the number of substitutions per site at first, second and third positions of codons in putative open reading frames, show that the variation of CMV-satRNAs does not follow a pattern typical of coding sequences, and indicates that preservation of the sequence of encoded products is not a constraint to evolution. On the other hand, when the observed variation was analyzed relative to a secondary structure model proposed for CMV-satRNAs, several lines of evidence indicated that the maintenance of the secondary structure is a constraint to evolution: the number of substitutions per site, the number of point insertions and deletions and the number of base substitutions that would disrupt base-pairing were significantly higher for unpaired than for base-paired positions. Also, compensatory mutations at base-paired positions occurred more frequently than expected from random. The results suggest that CMV-satRNAs are non-coding, functional RNAs whose biology would be determined by their direct interaction with components of the host and/or the helper virus.

Base Composition

Functional aspects of hemoglobin evolution in the mammals.

Comparative studies of red cells 2, 3 Diphosphoglycerate (DPG) and its effect on hemoglobin oxygen affinity from a taxonomically diverse set of mammals indicate two anomalous groups: members of the superfamilies Bovoidea (Actiodactyla) and Feloidea (Carnivora). In both taxa all of the individuals assayed had very low or unmeasurable quantities of DPG and red cell lysates with little, if any, DPG effect as measured by the change in oxygen affinity in the absence and presence of the phosphate. However, in both groups compensatory changes have occurred in hemoglobin structure and function so as to reduce the native oxygen affinity and thus cause them to resemble the hemoglobins of DPG-utilizing mammals as they occur in the setting of the red cell. We conclude that this parallelism of function is the result of convergent evolution.

Amino Acid Sequence

Long-term saline-alkaline selection rewires the growth-survival trade-off in Priestia megaterium.

Saline-alkaline soils impose persistent osmotic, ionic, pH, and nutrient stress on soil microorganisms, but the evolutionary routes by which beneficial bacteria adapt to such conditions remain poorly resolved. We performed adaptive laboratory evolution to examine the adaption of the plant growth-promoting rhizobacterium Priestia megaterium HA22 to long-term oligotrophic saline-alkaline selection. After 175 serial transfers, the evolved lineage proliferated stably at 40 g L-1 Na2SO4 at pH 10.0, whereas the wild-type strain failed to proliferate. Genome resequencing and allelic replacement revealed a 5-bp insertion in spo0A, the master sporulation regulator, as a major adaptive mutation. This mutation abolished sporulation; shortened the lag phase; and enhanced vegetative growth, nutrient uptake, and expression of tricarboxylic acid cycle and nitrogen metabolism gene under saline-alkaline stress. According to untargeted metabolomics, adaptation was accompanied by increased amino acid metabolism and aminoacyl-tRNA biosynthesis, with proline, isoleucine and pantothenic acid functionally promoting growth. A point mutation in ugpB enhanced glycerol-3-phosphate uptake, increased peptidoglycan and wall teichoic acid levels, and partially rescued the survival cost of the spo0A mutation. In greenhouse assays under combined saline-alkaline stress, the evolved strain increased soybean shoot dry weight and root dry weight by 56.08% and 27.02%, respectively. These results indicate that prolonged, predictable saline-alkaline selection can favor active growth rather than dormancy when compensatory cell envelope reinforcement buffers survival costs.

Adaptive laboratory evolution

Comparison of primary and secondary 26S rRNA structures in two Tetrahymena species: evidence for a strong evolutionary and structural constraint in expansion segments.

We have determined the nucleotide sequence of the 26S large subunit (LSU) rRNA genes for two Tetrahymena species, T. thermophila and T. pyriformis. The inferred rRNA sequences are presented in their most probable secondary structures based on compensatory mutations, energy, and conservation criteria. The majority of the nucleotide changes between the two Tetrahymena LSU rRNAs and the positions of a relatively large deletion and of the processing cleavage sites resulting in the generation of the hidden break are all located within the so-called divergent domains or expansion segments. These are regions within the common core of secondary structure where expansions have taken place during the evolution of the rRNA of higher eukaryotes. The dispensable nature of some of the expansion segments has been taken as evidence of their non-functionality. However, our data show that a considerable selective constraint has operated to preserve the secondary structure of these segments. Especially in the case of the D2 and D8 segments, the presence of a considerable number of compensatory base changes suggests that the secondary structure of these regions is of functional importance. Alternatively, these expansion segments may have maintained characteristic folding patterns because only such structures are being tolerated within otherwise functionally important regions.

Animals

[Hemodynamics of the eyes in diabetes mellitus].

The authors present evidence on the state of hemodynamics of the eye in patients suffering from diabetes mellitus on the basis of rheoophthalmographic and ophthalmoscopic studies with the use of the method of fluorescent angiography of the retina. The volume circulation of the eye proved to be connected with the intraophthalmic pressure and depended on the degree of affection of the eye vessels. Its changes were two-stage in character and depended on the state of the venous outflow. Diabetic retinopathy of the II stage served as the stage during which there was a sharp reduction of compensatory possibilities of hemodynamics, and further progress of vascular disturbances were observed. Rheoophthalmography permits to assess indirectly the degree of compensation of hemodynamic disturbances and can be used to asses clinical evolution of diabetic retinopathy.

Adolescent

Dinoflagellates in evolution. A molecular phylogenetic analysis of large subunit ribosomal RNA.

The sequence of the large subunit ribosomal RNA (LsuRNA) gene of the dinoflagellate Prorocentrum micans has been determined. The inferred rRNA sequence [3408 nucleotides (nt)] is presented in its most probable secondary structure based on compensatory mutations, energy, and conservation criteria. No introns have been found but a hidden break is present in the second variable domain, 690 nt from the 5' end, as judged by agarose gel electrophoresis and primer extension experiments. Prorocentrum micans LsuRNA length and G+C content are close to those of ciliates and yeast. The conserved portions of the molecule (1900 nt) have been aligned with corresponding sequences from various eukaryotes, including five protista, one metaphyta, and three metazoa. An extensive phylogenetic study was performed, comparing two phenetic methods (neighbor joining on difference matrix, and Fitch and Margoliash on Knuc values matrix) and one cladistic (parsimony). The three methods led to similar tree topologies, except for the emergence of yeast that groups with ciliates and dinoflagellates when phenetic methods are used, but emerges later in the most parsimonious tree. This discrepancy was checked by statistical analyses on reduced trees (limited to four species) inferred using parsimony and evolutionary parsimony methods. The data support the phenetic tree topologies and a close relationship between dinoflagellates, ciliates, and yeast.

Animals

Evolutionary adaptation to different thermal environments via transcriptional regulation.

Populations of the teleost fish Fundulus heteroclitus are subjected to the clinical variation in environmental temperatures that occurs along the eastern seacoast of North America. In concordance with this change in temperature is the clinal variation in the enzyme concentration of the heart-type lactate dehydrogenase (LDH-B; E.C.1.1.1.27). Previously we have shown that the compensating change in the LDH-B enzyme concentration is due to a change in the amount of LDH-B mRNA, but we did not define whether this was due to differences in mRNA stability or to differences in rate of transcription. The results presented here help clarify the molecular mechanism responsible for the variation in Ldh-B gene expression: the rate of transcription from the Ldh-B locus is significantly different between populations, and this difference is responsible for the compensatory change in LDH-B enzyme concentration.

Adaptation, Biological

Palatal epithelium of a monotreme and a marsupial.

The palatal epithelium of a monotreme, Tachyglossus aculeatus and a marsupial, Tarsipes spenserae were examined histologically and with the scanning electron microscope. Each animal possess keratinized palatal spines which although histologically similar, show significant differences in their external morphology. It is suggested that the spines in each case are highly differentiated filiform papillae which have developed as a compensatory mechanism of mastication, since both animals are in effect, edentulous. In the light of these findings and observations on the tongue of both animals, it is suggested that a degree of parallel evolution of the palate (as part of the masticatory apparatus) has occurred.

Animals